Siglec neutralizing antibodies

ABSTRACT

This invention relates to agents that bind human Siglecs having inhibitory activity in immune cells, and that neutralize the inhibitory activity of such Siglec. Such agents can be used for the treatment of cancers or infectious disease.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.16/082,959, filed Sep. 7, 2018, now U.S. Pat. No. 11,078,274, which isthe U.S. national stage application of International Patent ApplicationNo. PCT/EP2017/055364, filed Mar. 7, 2017, which claims the benefit ofU.S. Provisional Application No. 62/304,957 filed Mar. 8, 2016, thedisclosures of which are hereby incorporated by reference in theirentirety; including any drawings and sequence listing.

REFERENCE TO THE SEQUENCE LISTING

The present application is being filed along with a Sequence Listing inelectronic format. The Sequence Listing is provided as a file entitled“Sig792 PCT_ST25 txt”, created Feb. 22, 2017, which is 124 KB in size.The information in the electronic format of the Sequence Listing isincorporated herein by reference in its entirety.

FIELD OF THE INVENTION

This invention relates to agents that bind human Siglec proteins havinginhibitory activity in NK and/or other immune cells, and that neutralizethe inhibitory activity of such Siglec. Such agents can be used for thetreatment of cancers or infectious disease.

BACKGROUND OF THE INVENTION

NK cells are mononuclear cell that develop in the bone marrow fromlymphoid progenitors, and morphological features and biologicalproperties typically include the expression of the cluster determinants(CDs) CD16, CD56, and/or CD57; the absence of the alpha/beta orgamma/delta TCR complex on the cell surface; the ability to bind to andkill target cells that fail to express “self” major histocompatibilitycomplex (MHC)/human leukocyte antigen (HLA) proteins; and the ability tokill tumor cells or other diseased cells that express ligands foractivating NK receptors. NK cells are characterized by their ability tobind and kill several types of tumor cell lines without the need forprior immunization or activation. NK cells can also release solubleproteins and cytokines that exert a regulatory effect on the immunesystem; and can undergo multiple rounds of cell division and producedaughter cells with similar biologic properties as the parent cell.Normal, healthy cells are protected from lysis by NK cells.

Based on their biological properties, various therapeutic and vaccinestrategies have been proposed in the art that rely on a modulation of NKcells. However, NK cell activity is regulated by a complex mechanismthat involves both stimulating and inhibitory signals. Briefly, thelytic activity of NK cells is regulated by various cell surfacereceptors that transduce either positive or negative intracellularsignals upon interaction with ligands on the target cell. The balancebetween positive and negative signals transmitted via these receptorsdetermines whether or not a target cell is lysed (killed) by a NK cell.NK cell stimulatory signals can be mediated by Natural CytotoxicityReceptors (NCR) such as NKp30, NKp44, and NKp46; as well as NKG2Creceptors, NKG2D receptors, certain activating Killer Ig-like Receptors(KIRs), and other activating NK receptors (Lanier, Annual Review ofImmunology 2005; 23:225-74). NK cell inhibitory signals can be mediatedby receptors like CD94/NKG2-A, as well as certain inhibitory KIRs, whichrecognize major histocompatibility complex (MHC) class I-molecules(Wagtmann et al. (1995) Immunity 5:439-449). These inhibitory receptorsbind to polymorphic determinants of MHC class I molecules (including HLAclass I) present on other cells and inhibit NK cell-mediated lysis.

The lytic activity of NK cells can also be regulated by siglecpolypeptides. Siglecs (sialic-acid-binding immunoglobulin-like lectins)are a subset of I-type lectins that bind to sialoglycans and arepredominantly expressed on cells of the hematopoietic system in a mannerdependent on cell type and differentiation. Whereas sialic acid isubiquitously expressed, typically at the terminal position ofglycoproteins and lipids, only very specific, distinct sialoglycanstructures are recognized by individual Siglec receptors, depending onidentity and linkage to subterminal carbohydrate moieties. Siglecs haveonly low general affinity to the common mammalian sialoside structurescontaining the N-acetylneuraminic acid (Neu5Ac) α2-6 and α2-3 linkages.

Siglecs are generally divided into two groups, a first subset made up ofSiglec-1, -2, -4 and -15, and the CD33-related group of Siglecs whichincludes Siglec-3, -5, -6, -7, -8, -9, -10, -11, -12, -14 and -16. TheCD33-related Siglecs are characterized, inter alia, by low evolutionaryconservation and rapidly evolving sequence by multiple mechanisms.

Siglec-7 (CD328), a type 1 trans-membrane protein first cloned andcharacterized in 1999 by the Moretta group in Genoa, Italy, andbelonging to the human CD33-related Siglec receptors, is characterizedby a sialic acid binding N-terminal V-set Ig domain, two C2-set Igdomains and an intracytoplasmic region containing one immune-receptortyrosine based inhibitory motif (ITIM) and one ITIM-like motif. Siglec-7is constitutively expressed on NK cells, dendritic cells, monocytes andneutrophils. The extracellular domain of this receptor preferentiallybinds a (2,8)-linked disialic acids and branched a 2,6-sialyl residues,such as those displayed by ganglioside GD3.

Siglec-9 (CD329) was characterized in 2000 by the Varki group (see,e.g., Angata et al. J Biol Chem 2000; 275:22127-22135) and is expressedon monocytes, neutrophils, dendritic cells, CD34+ cells and NK cells.Siglec-9 (as well as Siglec-8) has been found to have differentialspecificity for sialoside ligands that contain both sialic acid andsulfate, with the position of the sulfate being an important determinantof specificity. Siglec-9 has been found to bind MUC16 that isoverexpressed on cancer cells. Like Siglec-7, Siglec 9 also contains asialic acid binding N-terminal V-set Ig domain, two C2-set Ig domainsand an intracytoplasmic region containing one immune-receptor tyrosinebased inhibitory motif (ITIM) and one ITIM-like motif. N-terminal V-setIg domain of human Siglec-9 shares an overall amino acid sequenceidentity of about 77% with N-terminal V-set Ig domain of human Siglec-7,and these two siglecs display different sialic acids bindingspecificities.

Binding assays have reported that, similar to Siglec-7, Siglec-9recognized sialic acid in either the α2,3- or α2,6-glycosidic linkage togalactose. Using a Siglec-9 specific mAb, Zhang et al. ((2000) J. Biol.Chem. Vol. 275, No. 29: 22121-22126) reported that Siglec-9 was found tobe expressed at high or intermediate levels by monocytes, neutrophils,and a minor population of CD16+, CD56− cells. However, weaker expressionwas observed on ˜50% of B cells and NK cells and minor subsets of CD8+ Tcells and CD4+ T cells. The authors concluded that despite their highdegree of sequence similarity, Siglec-7 and Siglec-9 have distinctexpression profiles.

Despite the interesting expression profile of Siglec-9 on NK and otherimmune cells, and the potential therapeutic interest in neutralizingSiglec-9, to date no candidate therapeutic agents that specificallyneutralize Siglec-9 have been advanced or proposed for therapeutic use.Engagement of Siglec-9 on cells of the myelomonocytic lineage bytumor-associated sialic acid ligands has been reported to inhibitimmunosurveillance and tumor cell killing by NK cells as well as byneutrophils, specialized granulocytes that recognize and directly killmicroorganisms (Laubli et al. (2014) Proc. Nat. Acad. Sci. USA 111(39):14211-14216). Carlin et al. ((2009) Blood 113: 3333-3336) reported thatmimicry of host sialylated glycans allows a bacterial pathogen to engageneutrophil Siglec-9 and dampen the innate immune response. Carlin et al.described anti-Siglec-9 antibody 191240 (R&D Systems, inc.) as bindingto the sialic acid binding site on Siglec-9 and inhibiting theinteraction with sialic acid. Carlin et al. further reported that unlikea non-blocking antibody (clone E10-286, BD Biosciences inc.), clone191240 enhanced the activation of neutrophils towards bacterial cells.Similarly, Laubli et al. (2014), supra, reported that the anti-Siglec-9antibody clone 191240 was able to enhance killing of tumor cells byneutrophils, compared to clone E10-286 that did not enhance killing oftumor cells by neutrophils.

Anti-Siglec-7 antibodies have been described in European Patent1238282B1 (Moretta et al) and Vitale et al. ((1999) Proc. Nat. Acad.Sci. 96(26):15091-96), referring to the murine anti-siglec-7 antibodyQA79, as well as in Falco et al. (1999) J. Exp. Med. 190:793-801 reportan anti-Siglec-7 antibody Z176.

Hudak et al. (2014) Nat. Chem. Biol. 10:69-77 reported that blockinganti-Siglec-7 antibodies inhibited the Siglec-7 mediated protection oftumor target cells from lysis by NK cells. However, when turning toSiglec-9, anti-Siglec-9 antibodies (clone 191240 was used) were not ableto inhibit the Siglec-9 mediated protection of tumor target cells fromlysis by NK cells purified from human donors (see, Hudak et al (2014)),despite the ability to enhance killing of tumor cells by neutrophils(see, Laubli et al. (2014). The bivalent binding antibody clone E10-286,reported in Laubli et al. (2014) as non-blocking and not enhancingkilling of tumor cells by neutrophils, also failed to inhibit theSiglec-9 mediated protection of tumor target cells from lysis by primaryNK cells (Jandus et al. (2014) J. Clin. Invest. 124(4): 1810-18020).

Despite the interest in Siglec-7 and -9, no therapeutic agents targetingthese receptors have been developed. There is therefore a need foragents that target these receptors for use in treating diseases such ascancer.

SUMMARY OF THE INVENTION

In one aspect, the present disclosure provides high affinity binderantibodies that act as potent neutralizers of human Siglecs, notably onNK cells in human individuals which express lower levels of cell surfaceSiglec compared to neutrophils and/or other cells, and that act asinhibitory cell surface receptors in effector lymphocytes (Siglec-7,Siglec-9).

In one embodiment, the disclosure provides Siglec inhibitors (e.g., aSiglec-9 expressed at the surface of a cell), including competitive andnon-competitive inhibitors of Siglec-9. The respective inhibitors haveparticularly high potency in neutralization of the inhibitory activityof a Siglec with or without substantially blocking the interactionbetween the Siglec and a sialic acid ligand thereof.

In one embodiment, the Siglec inhibitor is a protein comprising animmunoglobulin antigen binding domain that specifically binds to humanSiglec-9 protein, e.g. an antibody or antibody fragment, or a proteincomprising such. In one embodiment, the Siglec inhibitor specificallybinds to human Siglec-9 protein without binding to human Siglec-7 and/orother human Siglecs of Table 1 (exemplified by mAbsA, -B, -C, -D, -E and-F). In one embodiment, the Siglec inhibitor specifically binds to humanSiglec-9 protein and to human Siglec-7 protein, optionally furtherwithout binding to other human Siglecs of Table 1 (exemplified by mAbs4,-5, -6). In one embodiment, the Siglec inhibitor is an antibody orantibody fragment that specifically binds to human Siglec-9 protein, tohuman Siglec-7 protein and to human Siglec-12 protein, optionallyfurther without binding to other human Siglecs of Table 1 (exemplifiedby mAbs1, -2 and -3). In one embodiment, the Siglec inhibitor is anantibody or antibody fragment that is capable of bivalent binding to ahuman Siglec-9 protein (the inhibitor comprises two antigen bindingdomains that each are capable of binding to a human Siglec-9 protein).

In one embodiment, the disclosure provides an isolated antibody thatspecifically binds to a human Siglec-9 polypeptide and neutralizes theinhibitory activity of the Siglec-9 polypeptide, optionally wherein theantibody does not substantially block the interaction between theSiglec-9 polypeptide and a sialic acid ligand thereof (exemplified bymAbs1, 2 and 3, see Example 10), optionally wherein the antibody blocksthe interaction between the Siglec-9 polypeptide and a sialic acidligand thereof (exemplified by mAbsA, B and C, see Example 10).Optionally, the Siglec-9 polypeptide is expressed at the surface of acell, e.g., an effector lymphocyte, an NK cell, e.g., a primary NK cell,a CD56^(dim) NK cell from a human individual. In one embodiment, theantibody further binds to a human Siglec-7 polypeptide and neutralizesthe inhibitory activity of the Siglec-7 polypeptide, optionally furtherwherein the antibody does not substantially block the interactionbetween the Siglec-7 polypeptide and a sialic acid ligand thereof. Inanother embodiment, the antibody does not bind to a human Siglec-7polypeptide.

In one aspect, the present invention arises, inter alia, from thediscovery of antibodies that specifically bind human Siglec-9 and thatenhance the activity (e.g. cytotoxicity) of NK cells (e.g. primary NKcells) towards a sialic-acid ligand-bearing target cell. Unlike priorantibodies that can enhance cytotoxicity only in neutrophils, Siglectransfectants and/or other cells that express or are made to expresshigh levels of Siglec-9 at their cell surface, the antibodies describedherein are functional even in cells that express low levels of Siglec-9such as NK cells in a human (e.g. CD56^(dim) NK cells). The ability toenhance the cytotoxicity of such Siglec-9 low-expressing NK cells hasthe advantage of being able to additionally mobilize this population ofcells against disease target cells, e.g. tumor cells and/or bacterialcells.

In one embodiment, provided is an antibody or antibody fragment (or aprotein that comprises such a fragment) that specifically binds humanSiglec-9 and that enhances and/or restores the cytotoxicity of NK cells(primary NK cells) in a standard 4-hour in vitro cytotoxicity assay inwhich NK cells that express Siglec-9 are incubated with target cellsthat express a sialic acid ligand of Siglec-9. In one embodiment thetarget cells are labeled with ⁵¹Cr prior to addition of NK cells, andthen the killing (cytotoxicity) is estimated as proportional to therelease of ⁵¹Cr from the cells to the medium. Optionally, an assay canbe carried out according to the methods in the Examples herein, see,e.g. Example 8. In one embodiment, the antibody or antibody fragment iscapable of restoring cytotoxicity of NK cells that express Siglec-9 toat least the level observed with NK cells that do not express Siglec-9(e.g. as determined according to the methods of the Examples herein).

In any aspect herein, NK cells (e.g. primary NK cells) can be specifiedas being fresh NK cells purified from donors, optionally incubatedovernight at 37° C. before use. In any aspect herein, NK cells orprimary NK cells can be specified as being Siglec-9 expressing, e.g.,for use in assays the cells can be gated on Siglec-9 by flow cytometry.See, e.g. NK cells as described Example 8, herein.

In another embodiment, provided is an antibody or antibody fragment (ora protein that comprises such a fragment) that specifically binds humanSiglec-9 and that neutralizes the inhibitory activity of the Siglec-9polypeptide in a monocyte-derived dendritic cell (moDC). In oneembodiment, the moDC bear sialic acid ligands of Siglec-9 at theirsurface. In one embodiment, the moDC bear at their surface Siglec-9polypeptides that are engaged in cis-interactions with sialic acids. Inone embodiment, the antibody increases activation or signaling in amoDC. In one embodiment, the antibody neutralizes the inhibitoryactivity of the Siglec-9 polypeptide in a moDC bearing sialic acidligands of Siglec-9, wherein the moDC is a cell in which treatment ofthe moDC with neuramidase to remove sialic acid ligands results in alower EC₅₀ for antibody binding to the moDC.

In another aspect, the present invention arises, inter alia, from thediscovery of anti-Siglec antibodies that bind both Siglec-7 and Siglec-9polypeptides with comparable affinity. Such antibodies have advantageouspharmacological characteristics. As shown herein, NK cells can expressboth the inhibitory Siglec-7 and the inhibitory Siglec-9 protein, yetSiglec-7 and Siglec-9 can also have different expression profiles acrossdifferent cell populations. Furthermore, it has been shown that tumorcells can express the natural ligands (glycans) for Siglec-7 and forSiglec-9. Consequently, a therapeutic agent that inhibits of one Siglecbut not the other may not be maximally efficient in neutralizingSiglec-mediated restriction of the activity of NK and/or other immunecell populations. Inhibition of both Siglec 7 and 9 can therefore beadvantageous. However, Siglec-9 shares an overall amino acid sequenceidentity of only about 77% with N-terminal V-set Ig domain of humanSiglec-7. Moreover, these two siglecs display different sialic acidsbinding specificities suggesting structural differences in the regionthat is bound by sialic acid ligands.

In one aspect, the present disclosure provides high affinity binderantibodies that neutralize the inhibitory activity of Siglec-7 and/orSiglec-9 and are capable of specifically binding to the inhibitory humanSiglec-7 polypeptide and the human Siglec-9 polypeptide with comparableaffinity. The antibodies that bind Siglec-7 and Siglec-9 with comparableaffinity can, for example, in certain embodiments, have increasedability to block the interactions between each of the Siglecs and asialic acid ligand(s) thereof (exemplified by mAbs4, 5 and 6, seeExample 6). The antibodies that bind Siglec-9 can in other embodimentsbe characterized as neutralizing the inhibitory activity of Siglec-9,without substantially blocking the interactions between Siglec-9 and asialic acid ligand(s) thereof, particularly a sialic acid comprising aNeu5Aca2-3Galb1-4GlcNAcb structure (exemplified by mAbs1, 2 and 3, seeExample 9).

As shown herein, human Siglec-9 binds to both Sia1(Neu5Aca2-3Galb1-4GlcNAcb) and Sia2 (6′-Sialyllactose), while Siglec-7bind only to Sia2. Provided in one aspect are antibodies that arecapable blocking the interaction of such Siglec polypeptide(s) a sialicacid ligand of both Siglec-9 and Siglec-7, e.g., a Sia2 sialic acid. Inone embodiment, the sialic acid is a sialylated trisaccharide. In oneembodiment, the sialic acid comprises a 6′-Sialyllactose structure.

Provided in another aspect is an antibody that binds a human Siglec-9polypeptide and neutralizes the inhibitory activity thereof, wherein theantibody is capable of blocking the interaction of both Sia1(Neu5Aca2-3Galb1-4GlcNAcb) and Sia2 (6′-Sialyllactose) with a Siglec-9polypeptide (exemplified by mAbs1, 2 and 3, see Example 9).

In one embodiment, an antibody that is capable of binding Siglec-7 andSiglec-9 has an EC₅₀ for binding to human Siglec-7 polypeptide thatdiffers by less than 1-log from its EC₅₀ for binding to human Siglec-9polypeptide, as determined by flow cytometry for binding to cellsexpressing at their surface Siglec-7 or Siglec-9 (e.g., CHO cellstransfected with one of the respective Siglec but that do not expressthe other Siglec). In one embodiment, the antibody has an EC₅₀ forbinding to human Siglec-7 polypeptide and a human Siglec-9 polypeptidethat differs by no more than 0.5 log, 0.3 log, 0.2 log or 0.1 log, asdetermined by flow cytometry for binding to cells expressing at theirsurface Siglec-7 or Siglec-9. The cells expressing at their surfaceSiglec-7 or Siglec-9 can be characterized as expressing the respectivesiglec at comparable levels of expression.

In one embodiment, the antibodies further bind to non-human primateSiglec with a comparable affinity as for human Siglec-7 and/or -9. Inone embodiment, the antibody has an EC₅₀ for binding to human Siglec-7polypeptide, a human Siglec-9 polypeptide and a non-human primate Siglecthat differs by no more than 1-log, 0.5 log, 0.3 log, 0.2 log or 0.1log, as determined by flow cytometry for binding to cells expressing attheir surface Siglec-7 or Siglec-9 (e.g., CHO cells transfected with therespective Siglec).

In one embodiment, provided is an antibody that neutralizes the activityof human Siglec-9 and has an EC₅₀ for binding to a human Siglec-9polypeptide and a non-human primate Siglec that differs by no more than1-log, 0.5 log, 0.3 log, 0.2 log or 0.1 log, as determined by flowcytometry for binding to cells expressing at their surface the Siglec-9(e.g., CHO cells transfected with the Siglec).

In one embodiment, the antibody has a KD for binding affinity, asdetermined by, e.g., surface plasmon resonance (SPR) screening (such asby analysis with a BIAcore™ SPR analytical device), that differs by nomore than 10-fold, 5-fold, 3-fold or 2-fold for binding to a humanSiglec-7 polypeptide and to a human Siglec-9 polypeptide (and optionallyfurther a non-human primate Siglec).

In one embodiment, an antibody has a KD of about 1×10⁻⁸ M to about1×10⁻¹⁰ M, or about 1×10⁻⁹M to about 1×10⁻¹¹ M, for binding to a human ahuman Siglec-9 polypeptide (and optionally further a human Siglec-7polypeptide and/or non-human primate Siglec). In one embodiment, ananti-Siglec-9 antibody has a KD of about 1×10⁻⁸ M to about 1×10⁻¹⁰ M, orabout 1×10⁻⁹M to about 1×10⁻¹¹ M, for binding (e.g., monovalentaffinity) to a human a human Siglec-9 polypeptide, wherein the antibodydoes not have substantial binding to a human Siglec-7 polypeptide.

In one embodiment, the antibodies furthermore do not substantially bindany of human Siglecs-3, -5, -6, -8, -10, -11 and -12. In one embodiment,the antibodies furthermore do not substantially bind any of Siglecs-14and -16. In one embodiment, the antibodies furthermore do notsubstantially bind human Siglec-6. In one embodiment, the antibodiesfurthermore do not substantially bind human Siglec-12.

In any of the embodiments herein, the anti-Siglec antibodies can becharacterized by binding to polypeptides expressed on the surface of acell (e.g., an NK cell, a cell made to express Siglec-7 and/or Siglec-9,e.g., a recombinant CHO host cell made to express Siglec-7 and/orSiglec-9 at its surface, as shown in the Examples), and optionallyfurther wherein the antibody binds with high affinity as determined byflow cytometry. For example, an antibody can be characterized by anEC₅₀, as determined by flow cytometry, of no more than 5 μg/ml,optionally no more than 1 μg/ml, no more than 0.5 μg/ml, no more than0.2 μg/ml or no more than 0.1 μg/ml, for binding to primary NK cells(e.g., NK cells purified from a biological sample from a humanindividual or donor), optionally CD56^(dim) NK cells. EC50 can bedetermined, for example, according to the methods of Example 9, e.g., 4or more healthy human donors tested, stainings acquired on a BD FACSCanto II and analyzed using the FlowJo software, and EC₅₀ calculatedusing a 4-parameter logistic fit.

In another aspect, the present disclosure provides an antibody orantibody fragment (e.g. an antigen binding domain or a proteincomprising such), that specifically binds to a human Siglec-7 and/or -9polypeptide and is capable of a neutralizing the inhibitory activity ofsuch Siglec(s) in immune cells and capable of blocking the interactionof such Siglec polypeptide(s) with a sialic acid ligand thereof. In oneembodiment, the sialic acid is a sialylated trisaccharide. In oneembodiment, the sialic acid comprises a Neu5Aca2-3Galb1-4GlcNAcbstructure. In one embodiment, the sialic acid comprises a6′-Sialyllactose structure. In one embodiment, the antibody or antibodyfragment specifically binds to a human Siglec-7 and/or -9 polypeptideand is capable of a neutralizing the inhibitory activity of suchSiglec(s) in human NK cells (e.g. human primary NK cells; CD56^(dim) NKcells), in human monocytes, in human dendritic cells, in humanmacrophages (notably immunosuppressive or M2 macrophages), CD8 T cells,and/or in human neutrophils. In one embodiment, the antibody or antibodyfragment specifically binds to a human Siglec-7 and/or -9 polypeptideand is capable of a neutralizing the inhibitory activity of suchSiglec(s) in immunosuppressive macrophages (e.g. M2 macrophages) from ahuman donor, wherein the antibody or antibody fragment reduces theimmunosuppressive activity or capacity of the macrophages.

As discussed, human Siglec-9 binds to both Sia1(Neu5Aca2-3Galb1-4GlcNAcb) and Sia2 (6′-Sialyllactose), while Siglec-7bind only to Sia2. Provided in certain aspects are antibodies that arecapable blocking the interaction of a Siglec-9 polypeptide(s) with asialic acid that is a ligand of Siglec-9 but not Siglec-7, e.g., a Sia1sialic acid. In one embodiment, the sialic acid is a sialylatedtrisaccharide. In one embodiment, the sialic acid comprises aNeu5Aca2-3Galb1-4GlcNAcb structure. In one embodiment the antibody doesnot substantially block the interaction of a Siglec-7 polypeptide(s)with a sialic acid that is a ligand of Siglec-7 but not Siglec-9, e.g.,a 6′-Sialyllactose-containing sialic acid.

Fragments and derivatives of such antibodies are also provided. In oneembodiment, the antibody comprises an antigen-binding domain (e.g., asingle antigen binding domain, a domain made up of a heavy and a lightchain variable domain, etc.) capable of binding to the human Siglec-7polypeptide and/or human Siglec-9 polypeptide. In one embodiment, theantigen-binding domain binds human Siglec-9 polypeptide and not humanSiglec-7 polypeptide (exemplified by mAbsA, -B, -C, -D, -E and -F). Inone embodiment, the antigen-binding domain binds both human Siglec-9polypeptide and human Siglec-7 polypeptide (exemplified by mAbs1, -2,-3, -4, -5 and -6). In one embodiment, provided is a protein (e.g.antibody, multimeric and/or multispecific protein) or nucleic acidencoding such antigen binding domain.

In one embodiment, the neutralizing anti-Siglec antibody of thedisclosure relieves the inhibitory activity exerted by Siglec-7 and/or-9 in immune cells, enhancing the ability of lymphocytes to effectivelyrecognize and/or eliminate cancer cells that express sialic acid ligandsof Siglec-7 and/or sialic acid ligands of Siglec-9. The antibodies (orantibody fragments) reduce the ability of cancer cells to escape lysisdue to expression of one or the other types of ligand, and theytherefore enhance tumor surveillance by the immune system. In the NKcompartment, Siglec-9 is expressed primarily on CD56^(dim) NK cells,while siglec-7 is expressed on CD56^(dim) and CD56^(bright) NK cells.CD56^(dim) NK cells (CD56^(dim)CD16⁺KIR⁺) represent about 90% ofperipheral blood and spleen NK cells, express perforin and granzymes,and are the major cytotoxic subset, whereas CD56^(bright) NK cells(CD56^(bright)CD16^(dim/−)KIR⁻) constitute the majority of NK cells inlymph nodes and tonsils and, upon activation, primarily respond withcytokine production. In one embodiment, provided is an antibody orantibody fragment that specifically binds human Siglec-9 and relievesthe inhibitory activity exerted by Siglec-9 in human NK cells (e.g.human primary NK cells; CD56^(dim) NK cells), enhancing the ability ofthe NK cells to effectively recognize and/or eliminate cancer cells thatexpress sialic acid ligands of Siglec-9. In one embodiment, provided isan antibody or antibody fragment that specifically binds human Siglec-7and Siglec-9 and relieves the inhibitory activity exerted by Siglec-7and Siglec-9 in human NK cells (e.g. human primary NK cells; CD56^(dim)NK cells), enhancing the ability of the NK cells to effectivelyrecognize and/or eliminate cancer cells that express sialic acid ligandsof Siglec-7 and Siglec-9.

In one embodiment, an antibody of the disclosure can bind both Siglec-7and Siglec-9 and can neutralize both Siglec-7 and Siglec-9-mediatedinhibition of lymphocyte (e.g., NK cell, CD8+ T cell) cytotoxicity. Inone aspect, the antibody increases lymphocyte activation in the presenceof a target cell (e.g., a cell that expresses a ligand of Siglec-7and/or a ligand of Siglec-9, a tumor cell). In one embodiment, theantibody increases cytotoxicity of NK cells, as assessed in a standardin vitro cytotoxicity assay in which NK cells that express Siglec-9 arepurified from human donors and incubated with target cells that expressa sialic acid ligand of Siglec-9. In one embodiment, increasedactivation or neutralization of inhibition of cytotoxicity is assessedby increase in a marker of cytotoxicity/cytotoxic potential, e.g., CD107and/or CD137 expression (mobilization). In one embodiment, increasedactivation or neutralization of inhibition of cytotoxicity is assessedby increase in ⁵¹Cr release in a ⁵¹Cr release assay. The Siglec-7 maycomprise an amino acid sequence of SEQ ID NO: 1. The Siglec-9 maycomprise an amino acid sequence of SEQ ID NO: 2. In another embodiment,the Siglec-9 comprises an amino acid sequence of SEQ ID NO: 160.

In one embodiment, an antibody of the disclosure is capable of bindingto both a Siglec-9 polypeptide comprising the amino acid sequence of SEQID NO: 2 (bearing a lysine at position 100, representative of about 49%of the population) and to a Siglec-9 polypeptide comprising the aminoacid sequence of SEQ ID NO: 160 (bearing a glutamic acid at positioncorresponding to residue 100 of SEQ ID NO: 2), representative of about36% of the population). In any embodiment an antibody or antibodyfragment of the disclosure can be specified as being capable ofneutralizing the inhibitory activity of Siglec-9 in individuals whoexpress (or whose cells express) a Siglec-9 polypeptide comprising theamino acid sequence of SEQ ID NO: 2, as well as in individuals whoexpress (or whose cells express) a Siglec-9 polypeptide comprising theamino acid sequence of SEQ ID NO: 160.

In one embodiment, provided is an antibody or antibody fragment (or aprotein that comprises such fragment) that binds a human Siglec-9polypeptide and is capable of neutralizing the inhibitory activity ofboth a Siglec-9 polypeptide comprising the amino acid sequence of SEQ IDNO: 2 and a Siglec-9 polypeptide comprising the amino acid sequence ofSEQ ID NO: 160. In one embodiment, provided is an antibody or antibodyfragment (or a protein that comprises such fragment) that binds a humanSiglec-9 polypeptide and is capable of neutralizing the inhibitoryactivity of Siglec-9 polypeptide in NK cells that express a Siglec-9polypeptide comprising the amino acid sequence of SEQ ID NO: 2, and inNK cells that express a Siglec-9 polypeptide comprising the amino acidsequence of SEQ ID NO: 160. In one embodiment, the antibody increasescytotoxicity of NK cells, as assessed in a standard in vitrocytotoxicity assay in which NK cells that express the particularSiglec-9 are purified from human donors and incubated with target cellsthat express a sialic acid ligand of the Siglec-9.

In one aspect of any of the embodiments herein, the antibody is atetrameric (e.g., full length, F(ab)′2 fragment) antibody or antibodyfragment that bind an epitope present on the extracellular domain of aSiglec in bivalent fashion. For example, the antibody or antibodyfragment that binds a Siglec in bivalent fashion can comprise twoantigen binding domains that each are capable of binding a Siglec-9polypeptide, or that each are capable of binding to an epitope presenton both Siglec-7 and -9 polypeptides. In another aspect of any of theembodiments herein, the antibody binds to a Siglec in monovalent mannerand lacks agonist activity at each Siglec, e.g., Siglec-7 and/orSiglec-9. In one embodiment, the antibody that binds a Siglec inmonovalent manner is a Fab fragment. In any of the embodiments herein,the antibody binds to a Siglec in monovalent or bivalent manner is freeof agonist activity at the Siglec9. For therapeutic use, an antibody ispreferably a non-depleting antibody. Optionally the antibody comprisesan Fc domain capable of be bound by the human neonatal Fc receptor(FcRn) but which substantially lacks binding, via its Fc domain, to ahuman FcγR (e.g., CD16′ optionally one or more of, or each of, humanCD16A, CD16B, CD32A, CD32B and/or CD64 polypeptides). Optionally theantibody comprises and Fc domain of human IgG1, IgG2, IgG3 of IgG4isotype comprising an amino acid modification (e.g. one or moresubstitutions) that decrease the binding affinity of the antibody forone or more of, or each of, human CD16A, CD16B, CD32A, CD32B and/or CD64polypeptides.

In one embodiment, the antibody comprises one or more (e.g., two)antigen binding domain that binds to Siglec-9, optionally further toSiglec-7. In one specific embodiment, the antibody is a tetrameric,optionally full-length, antibody that comprises a two identical antigenbinding domains (optionally, two heavy and light chain variable regionpairs), and that binds and neutralizes the inhibitory activity ofSiglec-9, optionally further Siglec-7, comprises an Fc domain capable ofbe bound by the human neonatal Fc receptor (FcRn) and that substantiallylacks binding to a human FcγR (e.g., CD16; optionally one or more of, oreach of, human CD16A, CD16B, CD32A, CD32B and/or CD64 polypeptides).

In any of the embodiments herein, upon binding to a Siglec on a humanlymphocyte, the monoclonal antibody has the ability to enhance orreconstitute lysis of a target human cell bearing a sialic acid ligandof the Siglec on the target cell surface, and/or has the ability toincrease lymphocyte activation (e.g., as determined by an increase inCD107 and/or CD137 expression on a lymphocyte), when said target cellcomes into contact with said lymphocyte, e.g., an effector lymphocyte,an NK or a CD8+ T cell from a human individual, e.g. a CD56^(dim) NKcell. In one embodiment, provided is an antibody neutralizes a firstSiglec expressed by a first subset of lymphocytes (e.g. Siglec-9expressed on CD56^(dim) NK cells), and that neutralizes a second Siglecexpressed by a second subset of lymphocytes (Siglec-7 expressed onCD56^(bright) NK cells). The first and second subset of humanlymphocytes (e.g., NK cells, CD8+ T cells, monocytes, dendritic cells,macrophages, immunosuppressive or M2 macrophages) can for example becharacterized by different cell surface markers or different functionalproperties, or the ability to lyse or recognize (e.g., be activated by)different target cells. In one embodiment, the antibody reduces (blocks)binding of a Siglec to a sialoside ligand thereof (e.g., a ligandpresent on tumor cells).

In any of the embodiments herein, the sialoside or sialic acid ligand ofa Siglec is a natural ligand, e.g., a sialic acid ligand (a ligandcomprising a sialic acid) is known to bind to the Siglec polypeptide towhich the antibody binds. Sialic acids, a family of nine-carbon acidicmonosaccharides, are typically found to be terminating branches ofN-glycans, 0-glycans, and glycolipids. Siglecs are believed to recognizemany aspects of the sialic acid molecule, like the acid sialic linkagefrom the 2-position, the arrangements of sialic acids and their way ofpresentation. In any of the embodiments herein, the ligand of a Sigleccomprises mainly a 5-N-acetylneuraminic acid (Neu5Ac) derivative, andcan comprises other sialic acid derivatives, like 5-N-glycolylneuraminicacid (Neu5Gc) derivatives. In one embodiment, the ligand of Siglec-9and/or Siglec-7 is a sialic acid present on a glycoprotein (e.g., amucin) or a glycolipid. In one embodiment, the ligand of Siglec-7comprises a α2,8-linked disialic acid presented on b-seriesgangliosides, e.g., GD2, GD3 and GT1b. In one embodiment, the ligand ofSiglec-7 comprises an internally branched alpha2,6-linked disialicgangliosides, e.g., DSGb5. In one embodiment, the ligand of Siglec-9 isa ligand present on, or comprises, a mucin, e.g., MUC1. In oneembodiment, the ligand of Siglec-9 is a sialoglycan ligand that containsboth sialic acid and sulfate.

In one aspect, an antibody binds to a common determinant present on anextracellular domain of as first and a second human CD33-related Siglec.In one aspect of any embodiment herein, an antibody binds to adeterminant present on Siglec-9 but not on Siglec-7. In one aspect ofany embodiment herein, an antibody binds to a common determinant presenton Siglec-7 and on Siglec-9. Optionally, the determinant bound by anantibody is not present on one or more other Siglecs, e.g., one or moreof (or all of) Siglecs-3, -5, -6, -8, -10, -11 and -12.

In any of the embodiments herein, the antibody binds to an extracellulardomain of the Siglec. In certain of the embodiments herein, particularlywhere the antibody blocks the interaction between a Siglec and a sialicacid ligand thereof, the antibody may bind at least partially within ornear the sialic acid binding domain of the Siglec. In other embodimentsherein, particularly where the antibody does not block the interactionbetween a Siglec and a sialic acid ligand thereof, the antibody may bindoutside the sialic acid binding domain of the Siglec.

In any of the embodiments herein, upon binding to a Siglec on a humanlymphocyte (e.g., a primary NK cell), the monoclonal antibody has theability to reconstitute lysis of a target human cell bearing a sialicacid ligand of the Siglec on the target cell surface, when said targetcell comes into contact with said lymphocyte.

In any of the embodiments herein, the antibody has a KD (e.g. formonovalent binding, as determined according to the methods disclosed inthe Examples here) of less than 10⁻⁸ M, preferably less than 10⁻⁹ M forbinding to a Siglec polypeptide (e.g., human Siglec-7 and/or humanSiglec-9). Insofar as the Siglec-7 and -9 binding sites are believed togenerally masked at the cellular surface due to cis interactions withabundantly expressed low affinity sialic acids, trans interactions canoccur with antibodies expressing higher affinity than the ligands thatcompete with cis. In one embodiment, the neutralizing anti-Siglecantibody is capable of displacing the binding of a sialoside ligand to aSiglec (e.g., Siglec-7 and/or Siglec-9).

The invention also provides a human or humanized antibody or antibodyfragment, or a derivative thereof, which has any of the foregoingproperties, alone or in any suitable combination.

Provided in one aspect are monoclonal antibodies that compete forbinding to an epitope on Siglec-9 bound by mAbA, -B, -C, -D, -E and/or-F, (e.g., that competes for binding to an epitope on a Siglec-9polypeptide with an antibody having the heavy and light chain CDRs orvariable regions of any of mAbA, -B, -C, -D, -E and/or -F).

Provided in one aspect are monoclonal antibodies that compete forbinding to an epitope on Siglec-7 and/or Siglec-9 bound by mAbs1, -2,-3, -4, -5 and/or -6, (e.g., that competes for binding to an epitope ona Siglec-7 and/or Siglec-9 polypeptide with an antibody having the heavyand light chain CDRs or variable regions of any of mAbs1, -2, -3, -4, -5or -6).

In one aspect, the anti-Siglec antibodies have reduced binding to aSiglec-7 polypeptide having a mutation at residue N82, P83, A84, R85,A86 and/or V87 (e.g. the mutation as set forth in Table 3). In oneaspect, the anti-Siglec-7 antibodies have reduced binding to a Siglec-7polypeptide having a mutation at residue N81, D100, H102 and/or T103(e.g. the mutation as set forth in Table 3). In one aspect, theanti-Siglec-7 antibodies have reduced binding to a Siglec-7 polypeptidehaving a mutation at residue W88, E89, E90, R92 (e.g. the mutation asset forth in Table 3). Residue positions for mutations are withreference to the Siglec-7 polypeptide of SEQ ID NO: 1. Optionally, theantibody does not lose binding for one or more other mutant Siglec-7polypeptides of Table 3, e.g., one or more (or all of) mutants M6, M8,M15 or M16.

In one aspect, the anti-Siglec antibodies have reduced binding to aSiglec-9 polypeptide having a mutation at residue N78, P79, A80, R81,A82 and/or V83 (e.g. the mutation as set forth in Table 3). In oneaspect, the anti-Siglec-9 antibodies have reduced binding to a Siglec-9polypeptide having a mutation at residue N77, D96, H98 and/or T99 (e.g.the mutation as set forth in Table 3). In one aspect, the anti-Siglec-9antibodies have reduced binding to a Siglec-9 polypeptide having amutation at residue W84, E85, E86 and/or R88 (e.g. the mutation as setforth in Table 3). Residue positions for mutations are with reference tothe Siglec-9 polypeptide of SEQ ID NO: 2. Optionally, the antibody doesnot lose binding for one or more other mutant Siglec-9 polypeptides ofTable 3, e.g., one or more (or all of) mutants M6, M8, M15 or M16.

In one aspect, the anti-Siglec antibodies have reduced binding to aSiglec-9 polypeptide having a mutation at residue S47, H48, G49, W50,I51, Y52, P53 and/or G54 (e.g. the mutation as set forth in Table 3).Residue positions for mutations are with reference to the Siglec-9polypeptide of SEQ ID NO: 2. Optionally, the antibody does not losebinding for one or more other mutant Siglec-9 polypeptides of Table 3,e.g., mutants 9, 10 and/or 11, or one or more (or all of) mutants M7 orM8.

In one aspect, the anti-Siglec antibodies have reduced binding to aSiglec-9 polypeptide having a mutation at residue P55, H58, E122, G124,S125 and/or K127 (e.g. the mutation as set forth in Table 3). Residuepositions for mutations are with reference to the Siglec-9 polypeptideof SEQ ID NO: 2. Optionally, the antibody does not lose binding for oneor more other mutant Siglec-9 polypeptides of Table 3, e.g., mutants 9,10 and/or 11.

In one aspect, the anti-Siglec antibodies have reduced binding to aSiglec-9 polypeptide having a mutation at residue K131 and/or H132 (e.g.the mutation as set forth in Table 3). Residue positions for mutationsare with reference to the Siglec-9 polypeptide of SEQ ID NO: 2.Optionally, the antibody does not lose binding for one or more othermutant Siglec-9 polypeptides of Table 3, e.g., mutants 9, 10 and/or 11,or one or more (or all of) mutants M8 or M15.

In one aspect, the anti-Siglec antibodies have reduced binding to aSiglec-9 polypeptide having a mutation at residue R63, A66, N67, T68,D69, Q70 and/or D71 (e.g. the mutation as set forth in Table 3). Residuepositions for mutations are with reference to the Siglec-9 polypeptideof SEQ ID NO: 2. Optionally, the antibody does not lose binding for oneor more other mutant Siglec-9 polypeptides of Table 3, e.g., mutants 9,10 and/or 11, or one or more (or all of) mutants M6, M15 or M16.

In one embodiment, provided is antigen-binding compound that comprisesthe heavy and light chain CDR1, 2 and 3 of, or that binds the sameepitope and/or competes for binding to a Siglec-7 and/or Siglec-9polypeptide with, an antibody selected from the group consisting of:

(a) a monoclonal antibody comprising (i) a heavy chain comprising CDR 1,2 and 3 of the heavy chain variable region of SEQ ID NO: 3 and (ii) alight chain comprising CDR 1, 2 and 3 of the light chain variable regionof SEQ ID NO: 4;

(b) a monoclonal antibody comprising (i) a heavy chain comprising CDR 1,2 and 3 of the heavy chain variable region of SEQ ID NO: 5 and (ii) alight chain comprising CDR 1, 2 and 3 of the light chain variable regionof SEQ ID NO: 6;

(c) a monoclonal antibody comprising (i) a heavy chain comprising CDR 1,2 and 3 of the heavy chain variable region of SEQ ID NO: 7 and (ii) alight chain comprising CDR 1, 2 and 3 of the light chain variable regionof SEQ ID NO: 8;

(d) a monoclonal antibody comprising (i) a heavy chain comprising CDR 1,2 and 3 of the heavy chain variable region of SEQ ID NO: 9 and (ii) alight chain comprising CDR 1, 2 and 3 of the light chain variable regionof SEQ ID NO: 10;

(e) a monoclonal antibody comprising (i) a heavy chain comprising CDR 1,2 and 3 of the heavy chain variable region of SEQ ID NO: 11 and (ii) alight chain comprising CDR 1, 2 and 3 of the light chain variable regionof SEQ ID NO: 12;

(f) a monoclonal antibody comprising (i) a heavy chain comprising CDR 1,2 and 3 of the heavy chain variable region of SEQ ID NO: 13 and (ii) alight chain comprising CDR 1, 2 and 3 of the light chain variable regionof SEQ ID NO: 14;

(g) a monoclonal antibody comprising (i) a heavy chain comprising CDR 1,2 and 3 of the heavy chain variable region of SEQ ID NO: 15 and (ii) alight chain comprising CDR 1, 2 and 3 of the light chain variable regionof SEQ ID NO: 16;

(h) a monoclonal antibody comprising (i) a heavy chain comprising CDR 1,2 and 3 of the heavy chain variable region of SEQ ID NO: 17 and (ii) alight chain comprising CDR 1, 2 and 3 of the light chain variable regionof SEQ ID NO: 18;

(i) a monoclonal antibody comprising (i) a heavy chain comprising CDR 1,2 and 3 of the heavy chain variable region of SEQ ID NO: 19 and (ii) alight chain comprising CDR 1, 2 and 3 of the light chain variable regionof SEQ ID NO: 20;

(j) a monoclonal antibody comprising (i) a heavy chain comprising CDR 1,2 and 3 of the heavy chain variable region of SEQ ID NO: 21 and (ii) alight chain comprising CDR 1, 2 and 3 of the light chain variable regionof SEQ ID NO: 22;

(k) a monoclonal antibody comprising (i) a heavy chain comprising CDR 1,2 and 3 of the heavy chain variable region of SEQ ID NO: 23 and (ii) alight chain comprising CDR 1, 2 and 3 of the light chain variable regionof SEQ ID NO: 24; and

(l) a monoclonal antibody comprising (i) a heavy chain comprising CDR 1,2 and 3 of the heavy chain variable region of SEQ ID NO: 25 and (ii) alight chain comprising CDR 1, 2 and 3 of the light chain variable regionof SEQ ID NO: 26.

In one aspect of any of the embodiments of the invention, the antibodythat binds a Siglec-9 polypeptide may have a heavy chain having one, twoor three CDRs of the heavy chain of an antibody selected from the groupconsisting of antibody mAbA, -B, -C, -D, -E and —F; and a light chainhaving one, two or three CDRs of the light chain of the respectiveantibody selected from the group consisting of antibody mAbA, -B, -C,-D, -E and -F.

In one aspect, provided is an antigen binding domain or antibody thatbinds a human Siglec-9 polypeptide, comprising: a heavy chain CDR1comprising the amino acid sequence SYWMH (SEQ ID NO: 75); a heavy chainCDR2 comprising the amino acid sequence EINPSNGHTNYNEKFES (SEQ ID NO:78); a heavy chain CDR3 comprising the amino acid sequence GVESYDFDDALDY(SEQ ID NO: 80); a light chain CDR1 comprising the amino acid sequenceRASQDINNYLN (SEQ ID NO: 83); a light chain CDR2 comprising the aminoacid sequence YTSRLHS (SEQ ID NO: 57); a light chain CDR3 comprising theamino acid sequence QQGNTLPFT (SEQ ID NO: 86); and human heavy and lightchain framework sequences.

In one aspect, provided is an antigen binding domain or antibody thatbinds a human Siglec-9 polypeptide, comprising: a heavy chain CDR1comprising the amino acid sequence SYWMH (SEQ ID NO: 75); a heavy chainCDR2 comprising the amino acid sequence EINPSNGHTNYNEKFKT (SEQ ID NO:90); a heavy chain CDR3 comprising the amino acid sequence GVETYDFDDAMDY(SEQ ID NO: 92); a light chain CDR1 comprising the amino acid sequenceRASQDINNYLN (SEQ ID NO: 83); a light chain CDR2 comprising the aminoacid sequence FTSRLHS (SEQ ID NO: 95); a light chain CDR3 comprising theamino acid sequence QQGDTFPFT (SEQ ID NO: 96); and human heavy and lightchain framework sequences.

In one aspect, provided is an antigen binding domain or antibody thatbinds a human Siglec-9 polypeptide, comprising: a heavy chain CDR1comprising the amino acid sequence NYEMN (SEQ ID NO: 98); a heavy chainCDR2 comprising the amino acid sequence WINTYTGESTYADDFK (SEQ ID NO:101); a heavy chain CDR3 comprising the amino acid sequence DDYGRSYGFAY(SEQ ID NO: 103); a light chain CDR1 comprising the amino acid sequenceRASESVDSYGNSFMH (SEQ ID NO: 106); a light chain CDR2 comprising theamino acid sequence LASKLES (SEQ ID NO: 109); a light chain CDR3comprising the amino acid sequence HQNNEDPPWT (SEQ ID NO: 110); andhuman heavy and light chain framework sequences.

In one aspect, provided is an antigen binding domain or antibody thatbinds a human Siglec-9 polypeptide, comprising: a heavy chain CDR1comprising the amino acid sequence DYSMH (SEQ ID NO: 112); a heavy chainCDR2 comprising the amino acid sequence WIITETGEPTYADDFRG (SEQ ID NO:115); a heavy chain CDR3 comprising the amino acid sequence DFDGY (SEQID NO: 117); a light chain CDR1 comprising the amino acid sequenceRASENIYSYLA (SEQ ID NO: 119); a light chain CDR2 comprising the aminoacid sequence NAKTLTE (SEQ ID NO: 122); a light chain CDR3 comprisingthe amino acid sequence QHHYGFPWT (SEQ ID NO: 123); and human heavy andlight chain framework sequences.

In one aspect, provided is an antigen binding domain or antibody thatbinds a human Siglec-9 polypeptide, comprising: a heavy chain CDR1comprising the amino acid sequence TFGMH (SEQ ID NO: 125); a heavy chainCDR2 comprising the amino acid sequence YISSGSNAIYYADTVKG (SEQ ID NO:128); a heavy chain CDR3 comprising the amino acid sequence PGYGAWFAY(SEQ ID NO: 130); a light chain CDR1 comprising the amino acid sequenceRASSSVSSAYLH (SEQ ID NO: 133); a light chain CDR2 comprising the aminoacid sequence STSNLAS (SEQ ID NO: 136; a light chain CDR3 comprising theamino acid sequence QQYSAYPYT (SEQ ID NO: 137); and human heavy andlight chain framework sequences.

In one aspect, provided is an antigen binding domain or antibody thatbinds a human Siglec-9 polypeptide, comprising: a heavy chain CDR1comprising the amino acid sequence DYSMH (SEQ ID NO: 112); a heavy chainCDR2 comprising the amino acid sequence VISTYNGNTNYNQKFKG (SEQ ID NO:139); a heavy chain CDR3 comprising the amino acid sequence RGYYGSSSWFGY(SEQ ID NO: 141); a light chain CDR1 comprising the amino acid sequenceKASQNVGTDVA (SEQ ID NO: 144); a light chain CDR2 comprising the aminoacid sequence SASYRYS (SEQ ID NO: 147; a light chain CDR3 comprising theamino acid sequence QQYNSFPYT (SEQ ID NO: 148 and human heavy and lightchain framework sequences.

In one aspect of any of the embodiments of the invention, the antibodythat binds a Siglec-7 and a Siglec-9 polypeptide may have a heavy and/orlight chain having one, two or three CDRs of the respective heavy and/orlight chain of an antibody selected from the group consisting ofantibody mAbs1, -2, -3, -4, -5 and -6.

In one aspect of any of the embodiments of the invention, binding to aSiglec can be specified as being cellular Siglec, where the Siglec isexpressed at the surface of a cell, for example a native or modifiedcellular Siglec, a Siglec expressed by a recombinant host cell, a Siglecexpressed by an NK cell, a CD8 T cell, etc.

The invention also provides a nucleic acid encoding the human orhumanized antibody or antibody fragment having any of the foregoingproperties, a vector comprising such a nucleic acid, a cell comprisingsuch a vector, and a method of producing a human anti-Siglec antibody,comprising culturing such a cell under conditions suitable forexpression of the anti-Siglec antibody. The invention also relates tocompositions, such as pharmaceutically acceptable compositions and kits,comprising such proteins, nucleic acids, vectors, and/or cells andtypically one or more additional ingredients that can be activeingredients or inactive ingredients that promote formulation, delivery,stability, or other characteristics of the composition (e.g., variouscarriers). The invention further relates various new and useful methodsmaking and using such antibodies, nucleic acids, vectors, cells,organisms, and/or compositions, such as in the modulation ofSiglec-mediated biological activities, for example in the treatment ofdiseases related thereto, notably cancers and infectious disease.

In another aspect, provided is a method of producing an antibody whichneutralizes the inhibitory activity of Siglec-9, comprising:

-   -   (a) providing a plurality of antibodies that bind a Siglec-9        polypeptide,    -   (b) selecting antibodies (e.g., those of step of (a)) that        neutralize the inhibitory activity of a Siglec-9 polypeptide,        optionally in a primary human NK cell, e.g. a CD56^(dim) NK        cell, and    -   (c) selecting antibodies (e.g., those of step (b)) that do not        substantially block the interaction between a Siglec-9        polypeptide and a sialic acid ligand thereof. In one embodiment,        a Siglec-9 polypeptide is expressed at the surface of a cell,        e.g., a CHO cell, a lymphocyte, an NK cell.

In another aspect, provided is a method of producing an antibody whichneutralizes the inhibitory activity of Siglec-9, comprising:

-   -   (a) providing a plurality of antibodies that bind a Siglec-9        polypeptide,    -   (b) selecting antibodies (e.g., those of step of (a)) that        neutralize the inhibitory activity of a Siglec-9 polypeptide,        optionally in a primary human NK cell, e.g. a CD56^(d)′m NK        cell, and    -   (c) selecting antibodies (e.g., those of step (b)) that        substantially block the interaction between a Siglec-9        polypeptide and a sialic acid ligand thereof, optionally        selecting an antibody that blocks both a        Neu5Aca2-3Galb1-4GlcNAcb a 6′-Sialyllactose sialic acid ligand        of Siglec-9.

In another aspect, provided is a method of producing an antibody whichneutralizes the inhibitory activity of Siglec-7, comprising:

-   -   (a) providing a plurality of antibodies that bind a Siglec-7        polypeptide,    -   (b) selecting antibodies (e.g., those of step of (a)) that        neutralize the inhibitory activity of a Siglec-7 polypeptide,        and    -   (c) selecting antibodies (e.g., those of step (b)) that do not        substantially block the interaction between a Siglec-7        polypeptide and a sialic acid ligand thereof. In one embodiment,        a Siglec-7 polypeptide is expressed at the surface of a cell,        e.g., a CHO cell, a lymphocyte, an NK cell.

It will be appreciated that steps (b) and (c) in any of the abovemethods can be carried out in any desired order.

In another aspect, provided is a method of producing an antibody whichneutralizes the inhibitory activity of Siglec-9 and Siglec-7,comprising:

-   -   (a) providing a plurality of antibodies that bind a Siglec-9 and        a Siglec-7 polypeptide,    -   (b) selecting antibodies (e.g., those of step of (a)) that        neutralize the inhibitory activity of a Siglec-9 polypeptide and        a Siglec-7 polypeptide, optionally in primary human NK cells,        e.g. in CD56^(bright) and CD56^(dim) NK cell.

In one embodiment, selecting antibodies that neutralizes the inhibitoryactivity of a Siglec can comprise selecting antibodies that potentiateprimary NK cells (e.g. as purified NK cells obtained from human donors;for example according to the methods of Example 10).

In one embodiment, determining whether antibodies neutralize theinhibitory activity of any of the two different Siglec gene productscomprises assessing whether the antibody causes an increase in a markerof cytotoxicity (e.g., an increase in expression of CD107 and/or CD137)when lymphocytes expressing the Siglec(s) are brought into contact withtarget cells (e.g., that express ligands of the Siglec(s)). An increasein a marker of cytotoxicity (e.g., an increase in expression of CD107and/or CD137) indicates that the antibody is capable of neutralizing theinhibitory activity of the Siglec gene product(s).

The invention also provides an in vitro method for modulating theactivity of Siglec-7 and/or Siglec-9-expressing lymphocytes, optionallyNK cells and/or CD8+ T cells, the method comprising bringing lymphocytes(e.g. primary NK cells) expressing at their surface Siglec-7 and/orSiglec-9 into contact with an antibody that neutralizes the inhibitoryactivity of Siglec-7 and Siglec-9.

The invention also provides a method of potentiating and/or modulatingthe activity of lymphocytes (e.g., NK cells, CD8+ T cells) activity in asubject in need thereof, for example a method of potentiating NK cellactivity by modulating CD56^(dim) NK cells (the major cytotoxic subset)and optionally further CD56^(bright) NK cells (the majority of NK cellsin lymph nodes and tonsils and, upon activation, primarily respond withcytokine production), which method comprises administering to thesubject an effective amount of any of the foregoing compositions. In oneembodiment, the subject is a patient suffering from cancer. For example,the patient may be suffering from a hematopietic cancer, e.g., acutemyeloid leukaemia, chronic myeloid leukaemia, multiple myeloma, ornon-Hodgkin's lymphoma. Alternatively, the patient may be suffering froma solid tumor, e.g., colorectal cancer, renal cancer, ovarian cancer,lung cancer, breast cancer or malignant melanoma. In another embodiment,the subject is a patient suffering from an infectious disease.

These aspects are more fully described in, and additional aspects,features, and advantages will be apparent from, the description of theinvention provided herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows binding of anti-Siglec antibodies to NK cells. SiglecMFI:Mean of fluorescence intensity. A significant fraction (about 44%)of NK cells expressed both Siglec-7 and Siglec-9, suggesting that alarge proportion of NK cells can be inhibited by each of (or both of)these receptors, as a function of the glycan ligands present, forexample on tumor cells.

FIG. 2 shows representative results from flow cytometry for examples ofantibodies that bind to Siglec-7 but not Siglec-9 or cynomolgus Siglec(right panel), that bind to each of Siglec-7, Siglec-9 and cynomolgusSiglec (middle panel), and that bind to Siglec-9 but not Siglec-7 orcynomolgus Siglec (left panel).

FIG. 3 shows titration by flow cytometry binding of antibodies mAbA,mAbC and mAbD to moDC (left hand panel) and neuramidase-treated moDC(right hand panel), accompanied by their respective EC₅₀ values. TheEC₅₀ were highly enhanced (10 fold) after neuraminidase treatment,suggesting that Siglec-9 expressed on moDCs were engaged in cisinteraction with their sialic acid ligands before neuraminidasetreatment. However, the plateau phase level is not modified, suggestingthan the antibodies can bind all Siglec-9 (bound and unbound)conformations on cell surface and inhibits cis-interactions andsignalling in monoDCs, as well as in other cell types (e.g., monocytesand macrophages M1 and M2).

FIG. 4 shows dose dependent induction of an increase of YTS Siglec-9*cytotoxicity among Siglec-7 and -9 cross-reactive antibodies (FIG. 4B)and among the Siglec-9 monospecific (non-Siglec-7 binding) antibodies(FIG. 4A).

FIG. 5 shows the increase of primary NK cell cytotoxicity mediated byantibody mAbA, mAbC, mAbD, mAbE, and mAbF in two different human donors(donors D1 (left hand panel) and D2 (right hand panel)), in a classical⁵¹Cr release assay, using primary NK cells (as fresh NK cells purifiedfrom donors) and HT29 colorectal cancer cells.

FIG. 6 shows increase of % of Siglec-9-positive NK cells expressingCD137 mediated by several anti-Siglec-9 and anti Siglec-7/9 antibodiesmAbA, mAbB, mAbF, mAb6 and mAb4 in one human donor, in the presence ofHT29 tumor cells. The anti-Siglec-9 antibodies fully restoredcytotoxicity of Siglec-9-expressing primary human NK cells to the levelobserved in Siglec-9-negative primary human NK cells from the samedonor.

FIG. 7 shows that antibodies mAbA and mAb1 induce an increase ofSiglec-9-positive CD137+NK cells (%) (middle panel) but notSiglec-9-negative CD137+NK cells (%) (right hand panel). The % of NKexpressing CD137 in the absence of antibodies is shown in the left handpanel.

FIG. 8 shows binding of Siglec-9-Fc protein to Ramos cells in thepresence of antibodies (top panel). The anti-Siglec/9 mAbs mAbA, mAbB,mAbC, and mAbD each inhibited binding of Siglec-9-Fc protein to theRamos cells, while mAbE showed partial inhibition and mAbF did notinhibit binding. Binding of Siglec-9-Fc protein to K562 cells in thepresence of antibodies is shown in the bottom panel. The anti-Siglec/9mAbs mAbA, mAbB, mAbC and mAbD each inhibited binding of Siglec-9-Fcprotein to the Ramos cells, while both mAbE and mAbF showed partialinhibition.

FIGS. 9 and 10 show testing of blocking of the interaction betweenSiglec-7 and -9 and sialylated ligands by anti Siglec-7/9 antibodiesusing ELISA assays. FIG. 9 shows that mAbs 1, 2, 4, 5 and 6 blockedSiglec-7 interaction with Sia2, but mAb3 did not. FIG. 10 shows that allmAbs block the Siglec-9 interaction with Sia2, while mAb1, mAb2 and mAb3showed low ability to inhibit the Siglec-9 interaction with Sia1, andthus did not substantially block the Sia1 interaction.

FIGS. 11-14 show the human Siglec-9 protein. FIG. 11 shows the structureof the Siglec-9 N-terminal V-set Ig-like domain, with the residuessubstituted in Siglec-9 mutant M9, M10 and M11 shown in dark shading.FIG. 12 shows the structure of the Siglec-9 N-terminal V-set Ig-likedomain, with the residues substituted in Siglec-9 mutant M6 and M7 shownin dark shading. FIG. 13 shows the structure of the Siglec-9 N-terminalV-set Ig-like domain, with the residues substituted in Siglec-9 mutantM16 shown in dark shading. FIG. 14 shows the structure of the Siglec-9N-terminal V-set Ig-like domain, with the residues substituted inSiglec-9 mutant M8 shown in dark shading. In each of FIGS. 11-14, thesialic acid ligand binding site is shown in light shading.

DETAILED DESCRIPTION Definitions

As used in the specification, “a” or “an” may mean one or more. As usedin the claim(s), when used in conjunction with the word “comprising”,the words “a” or “an” may mean one or more than one. As used herein“another” may mean at least a second or more.

Where “comprising” is used, this can optionally be replaced by“consisting essentially of” or by “consisting of”.

Human Siglec-7 (shown in Genbank accession number NP_055200.1, theentire disclosure of which is incorporated herein by reference) is amember of the CD33-related Siglec family (Angata and Varki, Glycobiology10 (4), 431-438 (2000)). Human Siglec-7 comprises 467 amino acids,having the following amino acid sequence:

(SEQ ID NO: 1) mllllllpll wgrervegqk snrkdysltm gssvtvqegmcvhvrcsfsy pvdsqtdsdp vhgywfragn diswkapvatnnpawavqee trdrfhllgd pqtknctlsi rdarmsdagryffrmekgni kwnykydqls vnvtalthrp nilipgtlesgcfqnltcsv pwaceqgtpp miswmgtsvs plhpsttrssvltlipqpqh hgtsltcqvt lpgagvttnr tiqlnvsyppqnltvtvfqg egtastalgn ssslsvlegq slrlvcavdsnpparlswtw rsltlypsqp snplvlelqv hlgdegeftcragnslgsqh vslnlslqqe ytgkmrpvsg vllgavggagatalvflsfc vifivvrscr kksarpaadv gdigmkdantirgsasqgnl teswaddnpr hhglaahssg eereiqyaplsfhkgepqdl sgqeatnney seikipk.

Human Siglec-9 (shows in Genbank accession number NP055256.1 the entiredisclosure of which is incorporated herein by reference) is a member ofthe CD33-related Siglec family (Angata and Varki, J. Biol. Chem. 275(29), 22127-22135 (2000)). Human Siglec-9 comprises 463 amino acids,having the following amino acid sequence:

(SEQ ID NO: 2) mlllllpllw greraegqts klltmqssvt vqeglcvhvpcsfsypshgw iypgpvvhgy wfregantdq dapvatnnparavweetrdr fhllgdphtk nctlsirdar rsdagryffrmekgsikwny khhrlsvnvt althrpnili pgtlesgcpqnltcsvpwac eqgtppmisw igtsvspldp sttrssvltlipqpqdhgts ltcqvtfpga svttnktvhl nvsyppqnltmtvfqgdgtv stvlgngssl slpegqslrl vcavdavdsnpparlslswr gltlcpsqps npgvlelpwv hlrdaaeftcraqnplgsqq vylnvslqsk atsgvtqgvv ggagatalvflsfcvifvvv rscrkksarp aagvgdtgie danavrgsasqgpltepwae dsppdqpppa sarssvgege lqyaslsfqm vkpwdsrgqe atdteyseik ihr.

In the context of the present invention, “neutralize Siglec-mediatedinhibition of NK cell cytotoxicity”, ““neutralize Siglec-mediatedinhibition of T cell cytotoxicity” or “neutralize the inhibitoryactivity of a Siglec,”” refers to a process in which a Siglec (e.g.,Siglec-7, Siglec-9) is inhibited in its capacity to negatively affectintracellular processes leading to lymphocyte responses such as cytokinerelease and cytotoxic responses. This can be measured for example in astandard NK- or T-cell based cytotoxicity assay, in which the capacityof a therapeutic compound to stimulate killing of sialic-acid ligandpositive cells by Siglec positive lymphocytes is measured. In oneembodiment, an antibody preparation causes at least a 10% augmentationin the cytotoxicity of a Siglec-restricted lymphocyte, optionally atleast a 40% or 50% augmentation in lymphocyte cytotoxicity, oroptionally at least a 70% augmentation in NK cytotoxicity, and referringto the cytotoxicity assays described. In one embodiment, an antibodypreparation causes at least a 10% augmentation in cytokine release by aSiglec-restricted lymphocyte, optionally at least a 40% or 50%augmentation in cytokine release, or optionally at least a 70%augmentation in cytokine release, and referring to the cytotoxicityassays described. In one embodiment, an antibody preparation causes atleast a 10% augmentation in cell surface expression of a marker ofcytotoxicity (e.g., CD107 and/or CD137) by a Siglec-restrictedlymphocyte, optionally at least a 40% or 50% augmentation, or optionallyat least a 70% augmentation in cell surface expression of a marker ofcytotoxicity (e.g., CD107 and/or CD137).

The ability of an anti-Siglec antibody to “block” the binding of aSiglec molecule to a sialic acid ligand means that the antibody, in anassay using soluble or cell-surface associated Siglec and sialic acidmolecules, can detectably reduce the binding of a Siglec molecule to asialic acid molecule in a dose-dependent fashion, where the Siglecmolecule detectably binds to the sialic acid molecule in the absence ofthe antibody.

Whenever within this whole specification “treatment of cancer” or thelike is mentioned with reference to anti-Siglec binding agent (e.g.,antibody), there is meant: (a) method of treatment of cancer, saidmethod comprising the step of administering (for at least one treatment)an anti-Siglec binding agent, (preferably in a pharmaceuticallyacceptable carrier material) to an individual, a mammal, especially ahuman, in need of such treatment, in a dose that allows for thetreatment of cancer, (a therapeutically effective amount), preferably ina dose (amount) as specified herein; (b) the use of an anti-Siglecbinding agent for the treatment of cancer, or an anti-Siglec bindingagent, for use in said treatment (especially in a human); (c) the use ofan anti-Siglec binding agent for the manufacture of a pharmaceuticalpreparation for the treatment of cancer, a method of using ananti-Siglec binding agent for the manufacture of a pharmaceuticalpreparation for the treatment of cancer, comprising admixing ananti-Siglec binding agent with a pharmaceutically acceptable carrier, ora pharmaceutical preparation comprising an effective dose of ananti-Siglec binding agent that is appropriate for the treatment ofcancer; or (d) any combination of a), b), and c), in accordance with thesubject matter allowable for patenting in a country where thisapplication is filed.

As used herein, the term “antigen binding domain” refers to a domaincomprising a three-dimensional structure capable of immunospecificallybinding to an epitope. Thus, in one embodiment, said domain can comprisea hypervariable region, optionally a VH and/or VL domain of an antibodychain, optionally at least a VH domain. In another embodiment, thebinding domain may comprise at least one complementarity determiningregion (CDR) of an antibody chain. In another embodiment, the bindingdomain may comprise a polypeptide domain from a non-immunoglobulinscaffold.

The terms “antibody” or “immunoglobulin,” as used interchangeablyherein, include whole antibodies and any antigen binding fragment orsingle chains thereof. A typical antibody comprises at least two heavy(H) chains and two light (L) chains interconnected by disulfide bonds.Each heavy chain is comprised of a heavy chain variable region (V_(H))and a heavy chain constant region. The heavy chain constant region iscomprised of three domains, CH1, CH2, and CH3. Each light chain iscomprised of a light chain variable region (V_(L)) and a light chainconstant region. The light chain constant region is comprised of onedomain, CL. An exemplary immunoglobulin (antibody) structural unitcomprises a tetramer. Each tetramer is composed of two identical pairsof polypeptide chains, each pair having one “light” (about 25 kDa) andone “heavy” chain (about 50-70 kDa). The N-terminus of each chaindefines a variable region of about 100 to 110 or more amino acids thatis primarily responsible for antigen recognition. The terms variablelight chain (V_(L)) and variable heavy chain (V_(H)) refer to theselight and heavy chains respectively. The heavy-chain constant domainsthat correspond to the different classes of immunoglobulins are termed“alpha,” “delta,” “epsilon,” “gamma” and “mu,” respectively. Several ofthese are further divided into subclasses or isotypes, such as IgG1,IgG2, IgG3, IgG4, and the like. The subunit structures andthree-dimensional configurations of different classes of immunoglobulinsare well known. IgG are the exemplary classes of antibodies employedherein because they are the most common antibodies in the physiologicalsituation and because they are most easily made in a laboratory setting.Optionally the antibody is a monoclonal antibody. Particular examples ofantibodies are humanized, chimeric, human, or otherwise-human-suitableantibodies. “Antibodies” also includes any fragment or derivative of anyof the herein described antibodies.

A “cross-reactive” anti-Siglec antibody is an antibody that binds morethan one Siglec molecule with specificity and/or affinity. For example,a monoclonal antibody can be cross-reactive with Siglec-7 and Siglec-9.

The term “specifically binds to” means that an antibody can bindpreferably in a competitive binding assay to the binding partner, e.g.,Siglec-7, Siglec-9, as assessed using either recombinant forms of theproteins, epitopes therein, or native proteins present on the surface ofisolated target cells. Competitive binding assays and other methods fordetermining specific binding are further described below and are wellknown in the art.

When an antibody is said to “compete with” a particular monoclonalantibody, it means that the antibody competes with the monoclonalantibody in a binding assay using either recombinant Siglec molecules orsurface expressed Siglec molecules. For example, if a test antibodyreduces the binding of a reference antibody to a Siglec polypeptide orSiglec-expressing cell in a binding assay, the antibody is said to“compete” respectively with the reference antibody.

The term “affinity”, as used herein, means the strength of the bindingof an antibody to an epitope. The affinity of an antibody is given bythe dissociation constant Kd, defined as [Ab]×[Ag]/[Ab-Ag], where[Ab-Ag] is the molar concentration of the antibody-antigen complex, [Ab]is the molar concentration of the unbound antibody and [Ag] is the molarconcentration of the unbound antigen. The affinity constant K_(a) isdefined by 1/Kd. Methods for determining the affinity of mAbs can befound in Harlow, et al., Antibodies: A Laboratory Manual, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., 1988), Coligan etal., eds., Current Protocols in Immunology, Greene Publishing Assoc. andWiley Interscience, N.Y., (1992, 1993), and Muller, Meth. Enzymol.92:589-601 (1983), which references are entirely incorporated herein byreference. One standard method well known in the art for determining theaffinity of mAbs is the use of surface plasmon resonance (SPR) screening(such as by analysis with a BIAcore™ SPR analytical device).

Within the context herein a “determinant” designates a site ofinteraction or binding on a polypeptide.

The term “epitope” refers to an antigenic determinant, and is the areaor region on an antigen to which an antibody binds. A protein epitopemay comprise amino acid residues directly involved in the binding aswell as amino acid residues which are effectively blocked by thespecific antigen binding antibody or peptide, i.e., amino acid residueswithin the “footprint” of the antibody. It is the simplest form orsmallest structural area on a complex antigen molecule that can combinewith e.g., an antibody or a receptor. Epitopes can be linear orconformational/structural. The term “linear epitope” is defined as anepitope composed of amino acid residues that are contiguous on thelinear sequence of amino acids (primary structure). The term“conformational or structural epitope” is defined as an epitope composedof amino acid residues that are not all contiguous and thus representseparated parts of the linear sequence of amino acids that are broughtinto proximity to one another by folding of the molecule (secondary,tertiary and/or quaternary structures). A conformational epitope isdependent on the 3-dimensional structure. The term ‘conformational’ istherefore often used interchangeably with ‘structural’.

The term “deplete” or “depleting”, with respect to Siglec-expressingcells (e.g., Siglec-7 or Siglec-9 expressing lymphocytes) means aprocess, method, or compound that results in killing, elimination, lysisor induction of such killing, elimination or lysis, so as to negativelyaffect the number of such Siglec-expressing cells present in a sample orin a subject. “Non-depleting”, with reference to a process, method, orcompound means that the process, method, or compound is not depleting.

The term “agent” is used herein to denote a chemical compound, a mixtureof chemical compounds, a biological macromolecule, or an extract madefrom biological materials. The term “therapeutic agent” refers to anagent that has biological activity.

For the purposes herein, a “humanized” or “human” antibody refers to anantibody in which the constant and variable framework region of one ormore human immunoglobulins is fused with the binding region, e.g., theCDR, of an animal immunoglobulin. Such antibodies are designed tomaintain the binding specificity of the non-human antibody from whichthe binding regions are derived, but to avoid an immune reaction againstthe non-human antibody. Such antibodies can be obtained from transgenicmice or other animals that have been “engineered” to produce specifichuman antibodies in response to antigenic challenge (see, e.g., Green etal. (1994) Nature Genet 7:13; Lonberg et al. (1994) Nature 368:856;Taylor et al. (1994) Int Immun 6:579, the entire teachings of which areherein incorporated by reference). A fully human antibody also can beconstructed by genetic or chromosomal transfection methods, as well asphage display technology, all of which are known in the art (see, e.g.,McCafferty et al. (1990) Nature 348:552-553). Human antibodies may alsobe generated by in vitro activated B cells (see, e.g., U.S. Pat. Nos.5,567,610 and 5,229,275, which are incorporated in their entirety byreference).

A “chimeric antibody” is an antibody molecule in which (a) the constantregion, or a portion thereof, is altered, replaced or exchanged so thatthe antigen binding site (variable region) is linked to a constantregion of a different or altered class, effector function and/orspecies, or an entirely different molecule which confers new propertiesto the chimeric antibody, e.g., an enzyme, toxin, hormone, growthfactor, drug, etc.; or (b) the variable region, or a portion thereof, isaltered, replaced or exchanged with a variable region having a differentor altered antigen specificity.

The term “hypervariable region” when used herein refers to the aminoacid residues of an antibody that are responsible for antigen binding.The hypervariable region generally comprises amino acid residues from a“complementarity-determining region” or “CDR” (e.g., residues 24-34(L1), 50-56 (L2) and 89-97 (L3) in the light-chain variable domain and31-35 (H1), 50-65 (H2) and 95-102 (H3) in the heavy-chain variabledomain; Kabat et al. 1991) and/or those residues from a “hypervariableloop” (e.g., residues 26-32 (L1), 50-52 (L2) and 91-96 (L3) in thelight-chain variable domain and 26-32 (H1), 53-55 (H2) and 96-101 (H3)in the heavy-chain variable domain; Chothia and Lesk, J. Mol. Biol 1987;196:901-917), or a similar system for determining essential amino acidsresponsible for antigen binding. Typically, the numbering of amino acidresidues in this region is performed by the method described in Kabat etal., supra. Phrases such as “Kabat position”, “variable domain residuenumbering as in Kabat” and “according to Kabat” herein refer to thisnumbering system for heavy chain variable domains or light chainvariable domains. Using the Kabat numbering system, the actual linearamino acid sequence of a peptide may contain fewer or additional aminoacids corresponding to a shortening of, or insertion into, a FR or CDRof the variable domain. For example, a heavy chain variable domain mayinclude a single amino acid insert (residue 52a according to Kabat)after residue 52 of CDR H2 and inserted residues (e.g., residues 82a,82b, and 82c, etc. according to Kabat) after heavy chain FR residue 82.The Kabat numbering of residues may be determined for a given antibodyby alignment at regions of homology of the sequence of the antibody witha “standard” Kabat numbered sequence.

By “framework” or “FR” residues as used herein is meant the region of anantibody variable domain exclusive of those regions defined as CDRs.Each antibody variable domain framework can be further subdivided intothe contiguous regions separated by the CDRs (FR1, FR2, FR3 and FR4).

The terms “Fc domain,” “Fc portion,” and “Fc region” refer to aC-terminal fragment of an antibody heavy chain, e.g., from about aminoacid (aa) 230 to about aa 450 of human γ (gamma) heavy chain or itscounterpart sequence in other types of antibody heavy chains (e.g., α,δ, ϵ and μ for human antibodies), or a naturally occurring allotypethereof. Unless otherwise specified, the commonly accepted Kabat aminoacid numbering for immunoglobulins is used throughout this disclosure(see Kabat et al. (1991) Sequences of Protein of Immunological Interest,5th ed., United States Public Health Service, National Institute ofHealth, Bethesda, Md.).

The terms “isolated”, “purified” or “biologically pure” refer tomaterial that is substantially or essentially free from components whichnormally accompany it as found in its native state. Purity andhomogeneity are typically determined using analytical chemistrytechniques such as polyacrylamide gel electrophoresis or highperformance liquid chromatography. A protein that is the predominantspecies present in a preparation is substantially purified.

The terms “polypeptide,” “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidue is an artificial chemical mimetic of a corresponding naturallyoccurring amino acid, as well as to naturally occurring amino acidpolymers and non-naturally occurring amino acid polymer.

The term “recombinant” when used with reference, e.g., to a cell, ornucleic acid, protein, or vector, indicates that the cell, nucleic acid,protein or vector, has been modified by the introduction of aheterologous nucleic acid or protein or the alteration of a nativenucleic acid or protein, or that the cell is derived from a cell somodified. Thus, for example, recombinant cells express genes that arenot found within the native (nonrecombinant) form of the cell or expressnative genes that are otherwise abnormally expressed, under expressed ornot expressed at all.

Within the context herein, the term antibody that “binds” a polypeptideor epitope designates an antibody that binds said determinant withspecificity and/or affinity.

The term “identity” or “identical”, when used in a relationship betweenthe sequences of two or more polypeptides, refers to the degree ofsequence relatedness between polypeptides, as determined by the numberof matches between strings of two or more amino acid residues.“Identity” measures the percent of identical matches between the smallerof two or more sequences with gap alignments (if any) addressed by aparticular mathematical model or computer program (i.e., “algorithms”).Identity of related polypeptides can be readily calculated by knownmethods. Such methods include, but are not limited to, those describedin Computational Molecular Biology, Lesk, A. M., ed., Oxford UniversityPress, New York, 1988; Biocomputing: Informatics and Genome Projects,Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis ofSequence Data, Part 1, Griffin, A. M., and Griffin, H. G., eds., HumanaPress, New Jersey, 1994; Sequence Analysis in Molecular Biology, vonHeinje, G., Academic Press, 1987; Sequence Analysis Primer, Gribskov, M.and Devereux, J., eds., M. Stockton Press, New York, 1991; and Carilloet al., SIAM J. Applied Math. 48, 1073 (1988).

Methods for determining identity are designed to give the largest matchbetween the sequences tested. Methods of determining identity aredescribed in publicly available computer programs. Computer programmethods for determining identity between two sequences include the GCGprogram package, including GAP (Devereux et al., Nucl. Acid. Res. 12,387 (1984); Genetics Computer Group, University of Wisconsin, Madison,Wis.), BLASTP, BLASTN, and FASTA (Altschul et al., J. Mol. Biol. 215,403-410 (1990)). The BLASTX program is publicly available from theNational Center for Biotechnology Information (NCBI) and other sources(BLAST Manual, Altschul et al. NCB/NLM/NIH Bethesda, Md. 20894; Altschulet al., supra). The well-known Smith Waterman algorithm may also be usedto determine identity.

Production of Antibodies

The anti-Siglec agents useful for the treatment of disease (e.g. cancer,infectious disease) bind an extra-cellular portion of the human Siglec-9protein (and optionally further binding the human Siglec-7 protein, withor without further Siglec-12 binding) and reduces the inhibitoryactivity of the human Siglec expressed on the surface of a Siglecpositive immune cell. In one embodiment the agent inhibits the abilityof a sialic acid molecule to cause inhibitory signaling by a Siglec in aneutrophil, a dendritic cell, a macrophage, an M2 macrophage, an NK celland/or a CD8+ T cell.

In one embodiment, the anti-Siglec agent described herein can be used toincrease the cytotoxicity of NK cells and/or neutrophils in a human orfrom a human donor toward a target cell (e.g. a cancer cell) that bearsligands of the Siglec. NK cells and neutrophils are specializedgranulocytes that recognize and directly kill microorganisms and cancercells. Sialic acid expressing at the surface of tumor cells is shown toreduce the cytotoxicity of NK cells towards tumor cells. The antibodiescan be used to enhance NK cell cytotoxicity, for example to restore thelevel of cytotoxicity to substantially that observed in an NK cell orneutrophil that does not express at its surface the particular Siglec.

Sialic acids are also highly expressed on dendritic cells and have beendescribed to modulate several DC functions, including responsiveness toTLR stimulation. The blockade of sialic acid synthesis lowers theactivation threshold of moDCs for TLR stimulation and Siglec-E deletionenhanced dendritic cell responses to several microbial TLR ligands.Siglec-9 being the closest human orthologous member of Siglec-E in mice,the blocking anti-Siglec-9 antibodies may enhance dendritic cellactivation and modulate DC-T interactions. The modification of antigenswith sialic acids regulates the generation of antigen-specificregulatory T (Treg) cells and prevents formation of effector CD4+ andCD8+ T cells via dendritic cells. The phagocytic capacity of dendriticcells can also be improved by α2,6-sialic acid deficiency.

Siglec-7 and -9 are both expressed on type M1 and M2 macrophages, andthe knockdown of Siglec-9 has been described to modulate various surfaceexpression markers (e.g. CCR7 and CD200R) suggesting a modulation ofmacrophage functions by Siglec-9. Indeed, various Siglec-9 mutants(mutation in ITIM domain) were transfected in macrophage cell line anddemonstrated that Siglec-9 enhances the production of theanti-inflammatory cytokine IL-10. Binding of Siglec-9 with a solubleligand can also induce macrophages to display a tumor-associatedmacrophage-like phenotype, with increased expression of the checkpointligand PD-L1.

In one embodiment the agent competes with a sialic acid molecule inbinding to a Siglec, i.e., the agent blocks the interaction betweenSiglec and a sialic acid ligand thereof.

In one aspect of the invention, the agent is an antibody selected from afull-length antibody, an antibody fragment, and a synthetic orsemi-synthetic antibody-derived molecule.

In one aspect of the invention, the agent is an antibody selected from afully human antibody, a humanized antibody, and a chimeric antibody.

In one aspect of the invention, the agent is a fragment of an antibodyselected from IgA, an IgD, an IgG, an IgE and an IgM antibody.

In one aspect of the invention, the agent is a fragment of an antibodycomprising a constant domain selected from IgG1, IgG2, IgG3 and IgG4.

In one aspect of the invention, the agent is an antibody fragmentselected from a Fab fragment, a Fab′ fragment, a Fab′-SH fragment, aF(ab)2 fragment, a F(ab′)2 fragment, an Fv fragment, a Heavy chain Ig (allama or camel Ig), a V_(HH) fragment, a single domain FV, and asingle-chain antibody fragment.

In one aspect of the invention, the agent is a synthetic orsemisynthetic antibody-derived molecule selected from a scFV, a dsFV, aminibody, a diabody, a triabody, a kappa body, an IgNAR; and amultispecific antibody.

The present invention thus concerns antibodies and antigen bindingdomains (and polypeptides comprising the foregoing) that bind to Siglec.In one aspect, the antibody or antigen binding domain binds to Siglec-7and/or -9 with a binding affinity (e.g., KD) at least 100-fold lowerthan to a further human Siglec, e.g., Siglecs-3, -5, -6, -8, -10, -11and/or -12. In one aspect, the antibody or antigen binding domain bindsto Siglec-9 but not to Siglec-7; in one embodiment, the antibody binds ahuman Siglec-9 polypeptide with a binding affinity (e.g., KD) at least100-fold lower than to human Siglec-7 polypeptide. In another aspect,the antibody binds both a human Siglec-9 polypeptide and to humanSiglec-7 polypeptide with a binding affinity (e.g., KD) that does notdiffer by more than 1-log from one another, and wherein the bindingaffinities for said Siglec-7 and Siglec-9 are at least 100-fold lowerthan to a further human Siglec, e.g., Siglecs-3, -5, -6, -8, -10, -11and/or -12. Affinity can be determined for example by Surface PlasmonResonance, for binding to recombinant Siglec polypeptides.

In one aspect of the invention, the antibody is in purified or at leastpartially purified form. In one aspect of the invention, the antibody isin essentially isolated form.

The antibodies may be produced by a variety of techniques known in theart. Typically, they are produced by immunization of a non-human animal,preferably a mouse, with an immunogen comprising a Siglec polypeptide,preferably a human Siglec polypeptide. The Siglec polypeptide maycomprise the full length sequence of a human Siglec-9 and/or Siglec-7polypeptide, or a fragment or derivative thereof, typically animmunogenic fragment, i.e., a portion of the polypeptide comprising anepitope exposed on the surface of cells expressing a Siglec polypeptide.Such fragments typically contain at least about 7 consecutive aminoacids of the mature polypeptide sequence, even more preferably at leastabout 10 consecutive amino acids thereof. Fragments typically areessentially derived from the extra-cellular domain of the receptor. Inone embodiment, the immunogen comprises a wild-type human Siglecpolypeptide in a lipid membrane, typically at the surface of a cell. Ina specific embodiment, the immunogen comprises intact cells,particularly intact human cells, optionally treated or lysed. In anotherembodiment, the polypeptide is a recombinant Siglec polypeptide.

The step of immunizing a non-human mammal with an antigen may be carriedout in any manner well known in the art for stimulating the productionof antibodies in a mouse (see, for example, E. Harlow and D. Lane,Antibodies: A Laboratory Manual., Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y. (1988), the entire disclosure of which isherein incorporated by reference). The immunogen is suspended ordissolved in a buffer, optionally with an adjuvant, such as complete orincomplete Freund's adjuvant. Methods for determining the amount ofimmunogen, types of buffers and amounts of adjuvant are well known tothose of skill in the art and are not limiting in any way. Theseparameters may be different for different immunogens, but are easilyelucidated.

Similarly, the location and frequency of immunization sufficient tostimulate the production of antibodies is also well known in the art. Ina typical immunization protocol, the non-human animals are injectedintraperitoneally with antigen on day 1 and again about a week later.This is followed by recall injections of the antigen around day 20,optionally with an adjuvant such as incomplete Freund's adjuvant. Therecall injections are performed intravenously and may be repeated forseveral consecutive days. This is followed by a booster injection at day40, either intravenously or intraperitoneally, typically withoutadjuvant. This protocol results in the production of antigen-specificantibody-producing B cells after about 40 days. Other protocols may alsobe used as long as they result in the production of B cells expressingan antibody directed to the antigen used in immunization.

In an alternate embodiment, lymphocytes from a non-immunized non-humanmammal are isolated, grown in vitro, and then exposed to the immunogenin cell culture. The lymphocytes are then harvested and the fusion stepdescribed below is carried out.

For monoclonal antibodies, the next step is the isolation of splenocytesfrom the immunized non-human mammal and the subsequent fusion of thosesplenocytes with an immortalized cell in order to form anantibody-producing hybridoma. The isolation of splenocytes from anon-human mammal is well-known in the art and typically involvesremoving the spleen from an anesthetized non-human mammal, cutting itinto small pieces and squeezing the splenocytes from the splenic capsulethrough a nylon mesh of a cell strainer into an appropriate buffer so asto produce a single cell suspension. The cells are washed, centrifugedand resuspended in a buffer that lyses any red blood cells. The solutionis again centrifuged and remaining lymphocytes in the pellet are finallyresuspended in fresh buffer.

Once isolated and present in single cell suspension, the lymphocytes canbe fused to an immortal cell line. This is typically a mouse myelomacell line, although many other immortal cell lines useful for creatinghybridomas are known in the art. Murine myeloma lines include, but arenot limited to, those derived from MOPC-21 and MPC-11 mouse tumorsavailable from the Salk Institute Cell Distribution Center, San Diego,U.S.A, X63 Ag8653 and SP-2 cells available from the American TypeCulture Collection, Rockville, Md. U.S.A. The fusion is effected usingpolyethylene glycol or the like. The resulting hybridomas are then grownin selective media that contains one or more substances that inhibit thegrowth or survival of the unfused, parental myeloma cells. For example,if the parental myeloma cells lack the enzyme hypoxanthine guaninephosphoribosyl transferase (HGPRT or HPRT), the culture medium for thehybridomas typically will include hypoxanthine, aminopterin, andthymidine (HAT medium), which substances prevent the growth ofHGPRT-deficient cells.

Hybridomas are typically grown on a feeder layer of macrophages. Themacrophages are preferably from littermates of the non-human mammal usedto isolate splenocytes and are typically primed with incomplete Freund'sadjuvant or the like several days before plating the hybridomas. Fusionmethods are described in Goding, “Monoclonal Antibodies: Principles andPractice,” pp. 59-103 (Academic Press, 1986), the disclosure of which isherein incorporated by reference.

The cells are allowed to grow in the selection media for sufficient timefor colony formation and antibody production. This is usually betweenabout 7 and about 14 days.

The hybridoma colonies are then assayed for the production of antibodiesthat specifically bind to Siglec polypeptide gene products. The assay istypically a colorimetric ELISA-type assay, although any assay may beemployed that can be adapted to the wells that the hybridomas are grownin. Other assays include radioimmunoassays or fluorescence activatedcell sorting. The wells positive for the desired antibody production areexamined to determine if one or more distinct colonies are present. Ifmore than one colony is present, the cells may be re-cloned and grown toensure that only a single cell has given rise to the colony producingthe desired antibody. Typically, the antibodies will also be tested forthe ability to bind to Siglec polypeptides, e.g., Siglec-expressingcells.

Hybridomas that are confirmed to produce a monoclonal antibody can begrown up in larger amounts in an appropriate medium, such as DMEM orRPMI-1640. Alternatively, the hybridoma cells can be grown in vivo asascites tumors in an animal.

After sufficient growth to produce the desired monoclonal antibody, thegrowth media containing monoclonal antibody (or the ascites fluid) isseparated away from the cells and the monoclonal antibody presenttherein is purified. Purification is typically achieved by gelelectrophoresis, dialysis, chromatography using protein A or proteinG-Sepharose, or an anti-mouse Ig linked to a solid support such asagarose or Sepharose beads (all described, for example, in the AntibodyPurification Handbook, Biosciences, publication No. 18-1037-46, EditionAC, the disclosure of which is hereby incorporated by reference). Thebound antibody is typically eluted from protein A/protein G columns byusing low pH buffers (glycine or acetate buffers of pH 3.0 or less) withimmediate neutralization of antibody-containing fractions. Thesefractions are pooled, dialyzed, and concentrated as needed.

Positive wells with a single apparent colony are typically re-cloned andre-assayed to insure only one monoclonal antibody is being detected andproduced.

Antibodies may also be produced by selection of combinatorial librariesof immunoglobulins, as disclosed for instance in (Ward et al. Nature,341 (1989) p. 544, the entire disclosure of which is herein incorporatedby reference).

Antibodies can be titrated on Siglecs for the concentration required toachieve maximal binding to a Siglec polypeptide. “EC50” with respect tobinding to a Siglec polypeptide (or cell expressing such), refers to theefficient concentration of anti-Siglec antibody which produces 50% ofits maximum response or effect with respect to binding to a Siglecpolypeptide (or cell expressing such).

Once antibodies are identified that are capable of binding Siglec and/orhaving other desired properties, they will also typically be assessed,using standard methods including those described herein, for theirability to bind to other polypeptides, including other Siglecpolypeptides and/or unrelated polypeptides. Ideally, the antibodies onlybind with substantial affinity to Siglec, e.g., human Siglec-7 and/orhuman Siglec-9, and do not bind at a significant level to unrelatedpolypeptides, notably polypeptides other than CD33-related Siglecs, orSiglecs other than the desired Siglecs (e.g., Siglec-7 and/or Siglec-9).However, it will be appreciated that, as long as the affinity for Siglecis substantially greater (e.g., 5×, 10×, 50×, 100×, 500×, 1000×,10,000×, or more) than it is for other Siglecs and/or other, unrelatedpolypeptides), then the antibodies are suitable for use in the presentmethods.

The anti-Siglec antibodies can be prepared as non-depleting antibodiessuch that they have reduced, or substantially lack specific binding tohuman FCγ receptors. Such antibodies may comprise constant regions ofvarious heavy chains that are known not to bind, or to have low bindingaffinity for, FCγ receptors. One such example is a human IgG4 constantregion. Alternatively, antibody fragments that do not comprise constantregions, such as Fab or F(ab′)2 fragments, can be used to avoid Fcreceptor binding. Fc receptor binding can be assessed according tomethods known in the art, including for example testing binding of anantibody to Fc receptor protein in a BIACORE assay. Also, any antibodyisotype can be used in which the Fc portion is modified to minimize oreliminate binding to Fc receptors (see, e.g., WO03101485, the disclosureof which is herein incorporated by reference). Assays such as, e.g.,cell based assays, to assess Fc receptor binding are well known in theart, and are described in, e.g., WO03101485.

The DNA encoding an antibody that binds an epitope present on Siglecpolypeptides is isolated from the hybridoma and placed in an appropriateexpression vector for transfection into an appropriate host. The host isthen used for the recombinant production of the antibody, or variantsthereof, such as a humanized version of that monoclonal antibody, activefragments of the antibody, chimeric antibodies comprising the antigenrecognition portion of the antibody, or versions comprising a detectablemoiety.

DNA encoding a monoclonal antibodies can be readily isolated andsequenced using conventional procedures (e. g., by using oligonucleotideprobes that are capable of binding specifically to genes encoding theheavy and light chains of murine antibodies). Once isolated, the DNA canbe placed into expression vectors, which are then transfected into hostcells such as E. coli cells, simian COS cells, Chinese hamster ovary(CHO) cells, or myeloma cells that do not otherwise produceimmunoglobulin protein, to obtain the synthesis of monoclonal antibodiesin the recombinant host cells. As described elsewhere in the presentspecification, such DNA sequences can be modified for any of a largenumber of purposes, e.g., for humanizing antibodies, producing fragmentsor derivatives, or for modifying the sequence of the antibody, e.g., inthe antigen binding site in order to optimize the binding specificity ofthe antibody. Recombinant expression in bacteria of DNA encoding theantibody is well known in the art (see, for example, Skerra et al.,Curr. Opinion in Immunol., 5, pp. 256 (1993); and Pluckthun, Immunol.130, p. 151 (1992).

Within the context of this invention, a “common determinant” designatesa determinant or epitope that is shared by several gene products of thehuman inhibitory Siglec receptors, notably of the CD33-related Siglecs.An antibody can bind a common determinant shared by at least Siglec-7and Siglec-9. In one embodiment, the common determinant may optionallybe absent on one or more, or all of, the CD33-related Siglecs,particularly Siglecs-3, -5, -6, -8, -10, -11 and -12. In one embodimentthe common determinant is absent on Siglecs-3, -5, -6, -8, -10, -11 and-12.

The identification of one or more antibodies that bind(s) to siglecpolypeptides (e.g., Siglec-7 and/or Siglec-9, particularly substantiallyor essentially the same epitope as monoclonal antibody mAbsA, -B, -C,-D, -E or -F (Siglec-9 specific) or mAbs1, -2, -3, -4, -5 or -6(Siglec-7/9 specific), can be readily determined using any one of avariety of immunological screening assays in which antibody competitioncan be assessed. Many such assays are routinely practiced and are wellknown in the art (see, e. g., U.S. Pat. No. 5,660,827, which isincorporated herein by reference). It will be understood that actuallydetermining the epitope to which an antibody described herein binds isnot in any way required to identify an antibody that binds to the sameor substantially the same epitope as the monoclonal antibody describedherein.

For example, where the test antibodies to be examined are obtained fromdifferent source animals, or are even of a different Ig isotype, asimple competition assay may be employed in which the control (mAbA, -B,-C, -D, -E or -F or mAb1, -2, -3, -4, -5 or -6, for example) and testantibodies are admixed (or pre-adsorbed) and applied to a samplecontaining Siglec polypeptides. Protocols based upon western blottingand the use of BIACORE analysis are suitable for use in such competitionstudies.

In certain embodiments, one pre-mixes the control antibodies (mAbA, -B,-C, -D, -E or -F or mAb1, -2, -3, -4, -5 or -6, for example) withvarying amounts of the test antibodies (e.g., about 1:10 or about 1:100)for a period of time prior to applying to the Siglec antigen sample. Inother embodiments, the control and varying amounts of test antibodiescan simply be admixed during exposure to the Siglec antigen sample. Aslong as one can distinguish bound from free antibodies (e. g., by usingseparation or washing techniques to eliminate unbound antibodies) andmAbA, -B, -C, -D, -E, -F, -1, -2, -3, -4, -5 or -6 from the testantibodies (e. g., by using species-specific or isotype-specificsecondary antibodies or by specifically labeling mAbA, -B, -C, -D, -E,-F, -1, -2, -3, -4, -5 or -6 with a detectable label) one can determineif the test antibodies reduce the binding of mAbA, -B, -C, -D, -E, -F,-1, -2, -3, -4, -5 or -6 to the antigens, indicating that the testantibody competes for binding and/or recognizes a common binding site ona Siglec as mAb1, mAbA, -B, -C, -D, -E, -F, -1, -2, -3, -4, -5 or -6.The binding of the (labeled) control antibodies in the absence of acompletely irrelevant antibody can serve as the control high value. Thecontrol low value can be obtained by incubating the labeled (mAbA, -B,-C, -D, -E, -F, -1, -2, -3, -4, -5 or -6) antibodies with unlabelledantibodies of exactly the same type (mAbA, -B, -C, -D, -E, -F, -1, -2,-3, -4, -5 or -6), where competition would occur and reduce binding ofthe labeled antibodies. In a test assay, a significant reduction inlabeled antibody reactivity in the presence of a test antibody isindicative of a test antibody that recognizes substantially the sameregion on a Siglec, and that that “cross-reacts” or competes with thelabeled (mAbA, -B, —C, -D, -E, -F, -1, -2, -3, -4, -5 or -6) antibody.Any test antibody that reduces the binding of mAbA, -B, -C, -D, -E, -F,-1, -2, -3, -4, -5 or -6 to Siglec antigens by at least about 50%, suchas at least about 60%, or more preferably at least about 80% or 90% (e.g., about 65-100%), at any ratio of mAbA, -B, -C, -D, -E, -F, -1, -2,-3, -4, -5 or -6:test antibody between about 1:10 and about 1:100 isconsidered to be an antibody competes with the respective mAbA, -B, -C,-D, -E, -F, -1, -2, -3, -4, -5 or -6. Preferably, such test antibodywill reduce the binding of the respective mAbA, -B, -C, -D, -E, -F, -1,-2, -3, -4, -5 or -6 to the Siglec antigen by at least about 90% (e.g.,about 95%).

Competition can also be assessed by, for example, a flow cytometry test.In such a test, cells bearing one or more given Siglec polypeptide(s)can be incubated first with mAbA, -B, -C, -D, -E, -F, -1, -2, -3, -4, -5or -6, for example, and then with the test antibody labeled with afluorochrome or biotin. The antibody is said to compete with mAbA, -B,-C, -D, -E, -F, -1, -2, -3, -4, -5 or -6 if the binding obtained uponpreincubation with a saturating amount of mAbA, -B, -C, -D, -E, -F, -1,-2, -3, -4, -5 or -6 is about 80%, preferably about 50%, about 40% orless (e.g., about 30%, 20% or 10%) of the binding (as measured by meanof fluorescence) obtained by the antibody without preincubation with therespective mAbA, —B, -C, -D, -E, -F, -1, -2, -3, -4, -5 or -6.Alternatively, an antibody is said to compete with a mAbA, -B, -C, -D,-E, -F, -1, -2, -3, -4, -5 or -6 if the binding obtained with arespective labeled mAbA, -B, -C, -D, -E, -F, -1, -2, -3, -4, -5 or -6antibody (by a fluorochrome or biotin) on cells preincubated with asaturating amount of test antibody is about 80%, preferably about 50%,about 40%, or less (e. g., about 30%, 20% or 10%) of the bindingobtained without preincubation with the test antibody.

A simple competition assay in which a test antibody is pre-adsorbed andapplied at saturating concentration to a surface onto which a Siglecantigen is immobilized may also be employed. The surface in the simplecompetition assay is preferably a BIACORE chip (or other media suitablefor surface plasmon resonance analysis). The control antibody (e.g.,mAbA, -B, -C, -D, -E, -F, -1, -2, -3, -4, -5 or -6) is then brought intocontact with the surface at a Siglec-saturating concentration and theSiglec and surface binding of the control antibody is measured. Thisbinding of the control antibody is compared with the binding of thecontrol antibody to the Siglec-containing surface in the absence of testantibody. In a test assay, a significant reduction in binding of theSiglec-containing surface by the control antibody in the presence of atest antibody is indicative that the test antibody competes for bindingand thus may recognize the same region on a Siglec as the controlantibody such that the test antibody “cross-reacts” with the controlantibody. Any test antibody that reduces the binding of control (such asmAbA, -B, -C, -D, -E, -F, -1, -2, -3, -4, -5 or -6) antibody to a Siglecantigen by at least about 30% or more, preferably about 40%, can beconsidered to be an antibody that competes for binding to a Siglec as acontrol (e.g., a respective mAbA, -B, —C, -D, -E, -F, -1, -2, -3, -4, -5or -6). Preferably, such a test antibody will reduce the binding of thecontrol antibody (e.g., mAbA, -B, -C, -D, -E, -F, -1, -2, -3, -4, -5 or-6) to the Siglec antigen by at least about 50% (e. g., at least about60%, at least about 70%, or more). It will be appreciated that the orderof control and test antibodies can be reversed: that is, the controlantibody can be first bound to the surface and the test antibody isbrought into contact with the surface thereafter in a competition assay.Preferably, the antibody having higher affinity for the Siglec antigenis bound to the surface first, as it will be expected that the decreasein binding seen for the second antibody (assuming the antibodies arecross-reacting) will be of greater magnitude. Further examples of suchassays are provided in, e.g., Saunal (1995) J. Immunol. Methods 183:33-41, the disclosure of which is incorporated herein by reference.

Determination of whether an antibody binds within an epitope region canbe carried out in ways known to the person skilled in the art. As oneexample of such mapping/characterization methods, an epitope region foran anti-Siglec antibody may be determined by epitope “foot-printing”using chemical modification of the exposed amines/carboxyls in theSiglec protein. One specific example of such a foot-printing techniqueis the use of HXMS (hydrogen-deuterium exchange detected by massspectrometry) wherein a hydrogen/deuterium exchange of receptor andligand protein amide protons, binding, and back exchange occurs, whereinthe backbone amide groups participating in protein binding are protectedfrom back exchange and therefore will remain deuterated. Relevantregions can be identified at this point by peptic proteolysis, fastmicrobore high-performance liquid chromatography separation, and/orelectrospray ionization mass spectrometry. See, e. g., Ehring H,Analytical Biochemistry, Vol. 267 (2) pp. 252-259 (1999) Engen, J. R.and Smith, D. L. (2001) Anal. Chem. 73, 256A-265A. Another example of asuitable epitope identification technique is nuclear magnetic resonanceepitope mapping (NMR), where typically the position of the signals intwo-dimensional NMR spectra of the free antigen and the antigencomplexed with the antigen binding peptide, such as an antibody, arecompared. The antigen typically is selectively isotopically labeled with15N so that only signals corresponding to the antigen and no signalsfrom the antigen binding peptide are seen in the NMR-spectrum. Antigensignals originating from amino acids involved in the interaction withthe antigen binding peptide typically will shift position in thespectrum of the complex compared to the spectrum of the free antigen,and the amino acids involved in the binding can be identified that way.See, e. g., Ernst Schering Res Found Workshop. 2004; (44): 149-67; Huanget al., Journal of Molecular Biology, Vol. 281 (1) pp. 61-67 (1998); andSaito and Patterson, Methods. 1996 June; 9 (3): 516-24.

Epitope mapping/characterization also can be performed using massspectrometry methods. See, e.g., Downard, J Mass Spectrom. 2000 April;35 (4): 493-503 and Kiselar and Downard, Anal Chem. 1999 May 1; 71 (9):1792-1801. Protease digestion techniques also can be useful in thecontext of epitope mapping and identification. Antigenicdeterminant-relevant regions/sequences can be determined by proteasedigestion, e.g., by using trypsin in a ratio of about 1:50 to Siglec oro/n digestion at and pH 7-8, followed by mass spectrometry (MS) analysisfor peptide identification. The peptides protected from trypsin cleavageby the anti-Siglec binder can subsequently be identified by comparisonof samples subjected to trypsin digestion and samples incubated withantibody and then subjected to digestion by e.g., trypsin (therebyrevealing a footprint for the binder). Other enzymes like chymotrypsin,pepsin, etc., also or alternatively can be used in similar epitopecharacterization methods. Moreover, enzymatic digestion can provide aquick method for analyzing whether a potential antigenic determinantsequence is within a region of the Siglec polypeptide that is notsurface exposed and, accordingly, most likely not relevant in terms ofimmunogenicity/antigenicity.

Site-directed mutagenesis is another technique useful for elucidation ofa binding epitope. For example, in “alanine-scanning”, each residuewithin a protein segment is replaced with an alanine residue, and theconsequences for binding affinity measured. If the mutation leads to asignificant reduction in binding affinity, it is most likely involved inbinding. Monoclonal antibodies specific for structural epitopes (i.e.,antibodies which do not bind the unfolded protein) can be used to verifythat the alanine-replacement does not influence over-all fold of theprotein. See, e.g., Clackson and Wells, Science 1995; 267:383-386; andWells, Proc Natl Acad Sci USA 1996; 93:1-6.

Electron microscopy can also be used for epitope “foot-printing”. Forexample, Wang et al., Nature 1992; 355:275-278 used coordinatedapplication of cryoelectron micros-copy, three-dimensional imagereconstruction, and X-ray crystallography to determine the physicalfootprint of a Fab-fragment on the capsid surface of native cowpeamosaic virus.

Other forms of “label-free” assay for epitope evaluation include surfaceplasmon resonance (SPR, BIACORE) and reflectometric interferencespectroscopy (RifS). See, e.g., Fägerstam et al., Journal Of MolecularRecognition 1990; 3:208-14; Nice et al., J. Chroma-togr. 1993;646:159-168; Leipert et al., Angew. Chem. Int. Ed. 1998; 37:3308-3311;Kroger et al., Biosensors and Bioelectronics 2002; 17:937-944.

It should also be noted that an antibody binding the same orsubstantially the same epitope as an antibody of the invention can beidentified in one or more of the exemplary competition assays describedherein.

Cross-blocking assays can also be used to evaluate whether a testantibody affects the binding of the natural or non-natural sialic acidligand for human Siglec (e.g., Siglec-7 and/or Siglec-9). For example,to determine whether a humanized anti-Siglec antibody preparationreduces or blocks Siglec-7 interactions with sialic acid, the followingtest can be performed: A dose-range of anti-human Siglec-9 Fab isco-incubated 30 minutes at room temperature with the human Siglec-Fc(e.g., Siglec-7 Fc and/or Siglec-9 Fc) at a fixed dose, then added onsialic acid ligand expressing cell lines for 1 h. After washing cellstwo times in staining buffer, a PE-coupled goat anti-mouse IgG Fcfragment secondary antibodies diluted in staining buffer is added to thecells and plates are incubated for 30 additional minutes at 4° C. Cellsare washed two times and analyzed on an Accury C6 flow cytometerequipped with an HTFC plate reader. In the absence of test antibodies,the Siglec-Fc binds to the cells. In the presence of an antibodypreparation pre-incubated with Siglec-Fc (e.g., Siglec-7 Fc and/orSiglec-9 Fc) that blocks Siglec-binding to sialic acid, there is areduced binding of Siglec-Fc to the cells. However, it will beappreciated that reconstitution of NK cell lytic activity toward sialicacid ligand-expressing target cells can be assessed directly without theneed to assess blockade of the Siglec-sialic acid ligand interaction.

Optionally, antibodies of the disclosure can be specified to beantibodies other than any one or more of antibodies E10-286 (BDBiosciences Corp.), QA79 disclosed in European Patent 1238282B1 (Morettaet al., Universita degli Studi di Genova), or Z176 referenced in Falcoet al. (1999) J. Exp. Med. 190:793-801, or derivatives of the foregoing,e.g., that comprise the antigen binding region or heavy and/or lightchain CDRs, in whole or in part. Optionally, antibodies of thedisclosure can be specified to be antibodies other than any one or moreof antibodies 3A11, 1H9 and 2B4 disclosed in PCT application no.PCT/EP2015/070550 filed 9 Sep. 2015 (Innate Pharma). In otherembodiments, the above-mentioned antibodies may, depending on the natureof the antibody, be modified so as to have the characteristics of theantibodies of the present disclosure.

Provided herein are antibodies that bind the extracellular domain, e.g.,the N-terminal V-set domain or the Ig-like C2-type domain 1 or 2 ofhuman Siglec-9, for example antibodies that bind the epitopes shown inthe Examples herein.

In one aspect, the antibodies bind substantially the same epitope asantibody mAb1, -2, -3, -4, -5, -6, -A, -B, -C, -D, -E or -F. In oneembodiment, the antibodies bind to an epitope of Siglec-9 and/orSiglec-7 that at least partially overlaps with, or includes at least oneresidue in, the epitope bound by antibody mAb1, -2, -3, -4, -5, -6, -A,-B, -C, -D, -E or -F. The residues bound by the antibody can bespecified as being present on the surface of the of the Siglec-9 and/orSiglec-7 polypeptide, e.g. in a Siglec-9 or Siglec-7 polypeptideexpressed on the surface of a cell. The amino acid residues on Siglec-9and/or Siglec-7 bound by the antibody can for example be selected fromthe group consisting of the residues listed in Table 3.

Binding of anti-Siglec antibody to cells transfected with Siglec-9mutants can be measured and compared to the ability of anti-Siglecantibody to bind wild-type Siglec-9 polypeptide (e.g., SEQ ID NO: 2).For antibodies that additionally bind Siglec-7, binding of anti-Siglecantibody can additionally or alternatively be conducted using cellstransfected with Siglec-7 mutants (e.g. of Table 3) and compared to theability of anti-Siglec antibody to bind wild-type Siglec-7 polypeptide(e.g., SEQ ID NO: 1). A reduction in binding between an anti-Siglecantibody and a mutant Siglec-9 and/or Siglec-7 polypeptide (e.g., amutant Siglec-9 or Siglec-7 of Table 3) means that there is a reductionin binding affinity (e.g., as measured by known methods such FACStesting of cells expressing a particular mutant, or by Biacore™ (SPR)testing of binding to mutant polypeptides) and/or a reduction in thetotal binding capacity of the anti-Siglec antibody (e.g., as evidencedby a decrease in Bmax in a plot of anti-Siglec antibody concentrationversus polypeptide concentration). A significant reduction in bindingindicates that the mutated residue is directly involved in binding tothe anti-Siglec antibody or is in close proximity to the binding proteinwhen the anti-Siglec antibody is bound to Siglec-9.

In some embodiments, a significant reduction in binding means that thebinding affinity and/or capacity between an anti-Siglec antibody and amutant Siglec-9 polypeptide is reduced by greater than 40%, greater than50%, greater than 55%, greater than 60%, greater than 65%, greater than70%, greater than 75%, greater than 80%, greater than 85%, greater than90% or greater than 95% relative to binding between the antibody and awild type Siglec-9 polypeptide. In certain embodiments, binding isreduced below detectable limits. In some embodiments, a significantreduction in binding is evidenced when binding of an anti-Siglecantibody to a mutant Siglec-9 polypeptide is less than 50% (e.g., lessthan 45%, 40%, 35%, 30%, 25%, 20%, 15% or 10%) of the binding observedbetween the anti-Siglec antibody and a wild-type Siglec-9 polypeptide.

In some embodiments, anti-Siglec antibodies are provided that exhibitsignificantly lower binding for a mutant Siglec-9 and/or Siglec-7polypeptide in which a residue in a segment comprising an amino acidresidue bound by antibody mAb1, -2, -3, -A, -B, -C, -D, -E or -F issubstituted with a different amino acid. In one embodiment, the mutantis a mutant selected from mutants M6, M8, M9, M10, M11, M15 and M16 ofTable 3, compared to binding to a wild-type Siglec polypeptide (e.g. theSiglec-9 polypeptide of SEQ ID NO: 2). In one embodiment, the mutant isa mutant selected from mutants M6, M8, M9, M10, M11, M15 and M16 ofTable 3, compared to binding to a wild-type Siglec-7 polypeptide (e.g.the polypeptide of SEQ ID NO: 1).

In one aspect, the anti-Siglec antibodies bind an epitope on humanSiglec-9 comprising one, two, three, four, five or six of the residuesselected from the group consisting of N78, P79, A80, R81, A82 and/or V83(with reference to SEQ ID NO: 2).

In one aspect, the anti-Siglec antibodies bind an epitope on humanSiglec-9 comprising one, two, three or four of the residues selectedfrom the group consisting of N77, D96, H98 and/or T99 (with reference toSEQ ID NO: 2).

In one aspect, the anti-Siglec antibodies bind an epitope on humanSiglec-9 comprising one, two, three or four of the residues selectedfrom the group consisting of W84, E85, E86 and/or R88 (with reference toSEQ ID NO: 2).

In one aspect, the anti-Siglec antibodies bind an epitope on humanSiglec-9 comprising one, two, three, four, five, six, seven or eight ofthe residues selected from the group consisting of S47, H48, G49, W50,I51, Y52, P53 and/or G54 (with reference to SEQ ID NO: 2).

In one aspect, the anti-Siglec antibodies bind an epitope on humanSiglec-9 comprising one or both of the residues K131 and/or H132 (withreference to SEQ ID NO: 2).

In one aspect, the anti-Siglec antibodies bind an epitope on humanSiglec-9 comprising one, two, three, four, five, six or seven of theresidues selected from the group consisting of R63, A66, N67, T68, D69,Q70 and/or D71 (with reference to SEQ ID NO: 2).

In one aspect, the anti-Siglec antibodies bind an epitope on humanSiglec-9 comprising one, two, three, four, five or six of the residuesselected from the group consisting of P55, H58, E122, G124, S125 and/orK127 (with reference to SEQ ID NO: 2).

In one aspect, the anti-Siglec antibodies bind an epitope on humanSiglec-7 comprising one, two, three, four, five or six of the residuesselected from the group consisting of N82, P83, A84, R85, A86 and/or V87(with reference to SEQ ID NO: 1).

In one aspect, the anti-Siglec antibodies bind an epitope on humanSiglec-7 comprising one, two, three or four of the residues selectedfrom the group consisting of N81, D100, H102 and/or T103 (with referenceto SEQ ID NO: 1).

In one aspect, the anti-Siglec antibodies bind an epitope on humanSiglec-7 comprising one, two, three or four of the residues selectedfrom the group consisting of W88, E89, E90, R92 (with reference to SEQID NO: 1).

Once an antigen-binding compound having the desired binding for Siglecsis obtained it may be assessed for its ability to inhibit Siglec (e.g.,Siglec-7 and/or Siglec-9). For example, if an anti-Siglec antibodyreduces or blocks Siglec activation induced by a sialic acid ligand(e.g., as present on a cell), it can increase the cytotoxicity ofSiglec-restricted lymphocytes. This can be evaluated by a typicalcytotoxicity assay, examples of which are described below.

The ability of an antibody to reduce Siglec-mediated signaling can betested in a standard 4-hour in vitro cytotoxicity assay using, e.g., NKcells that express Siglec-7 or Siglec-9, and target cells that express asialic acid ligand of the respective Siglec. Such NK cells do notefficiently kill targets that express the sialic acid ligand becauseSiglec-7 or -9 recognizes the sialic acid ligand, leading to initiationand propagation of inhibitory signaling that preventslymphocyte-mediated cytolysis. Such an assay can be carried outaccording to the methods in the Examples herein, see, e.g. Example 8,using primary NK cells, as fresh NK cells purified from donors,incubated overnight at 37° C. before use. Such an in vitro cytotoxicityassay can be carried out by standard methods that are well known in theart, as described for example in Coligan et al., eds., Current Protocolsin Immunology, Greene Publishing Assoc. and Wiley Interscience, N.Y.,(1992, 1993). The target cells are labeled with ⁵¹Cr prior to additionof NK cells, and then the killing is estimated as proportional to therelease of ⁵¹Cr from the cells to the medium, as a result of killing.The addition of an antibody that prevents Siglec-7 and/or -9 frombinding to the sialic acid ligand results in prevention of theinitiation and propagation of inhibitory signaling via the Siglec.Therefore, addition of such agents results in increases inlymphocyte-mediated killing of the target cells. This step therebyidentifies agents that prevent Siglec-7 or -9-induced negative signalingby, e.g., blocking ligand binding. In a particular ⁵¹Cr-releasecytotoxicity assay, Siglec-7 or -9-expressing NK effector-cells can killsialic acid ligand-negative target cells (e.g., cells treated withsialidase), but less well sialic acid ligand-expressing control cells.Thus, NK effector cells kill less efficiently sialic acid ligandpositive cells due to sialic acid-induced inhibitory signaling via theparticular Siglec. When NK cells are pre-incubated with blockinganti-Siglec antibodies in such a ⁵¹Cr-release cytotoxicity assay, sialicacid ligand-expressing cells are more efficiently killed, in anantibody-concentration-dependent fashion. The assay can be carried outseparately for each Siglec, e.g., Siglec-7 and Siglec-9.

The inhibitory activity (i.e., cytotoxicity enhancing potential) of anantibody can also be assessed in any of a number of other ways, e.g., byits effect on intracellular free calcium as described, e.g., in Sivoriet al., J. Exp. Med. 1997; 186:1129-1136, the disclosure of which isherein incorporated by reference, or by the effect on markers of NK cellcytotoxicity activation, such as degranulation marker CD107 or CD137expression. NK, T, or NKT cell activity can also be assessed using anycell based cytotoxicity assays, e.g., measuring any other parameter toassess the ability of the antibody to stimulate NK cells to kill targetcells such as P815, K562 cells, or appropriate tumor cells as disclosedin Sivori et al., J. Exp. Med. 1997; 186:1129-1136; Vitale et al., J.Exp. Med. 1998; 187:2065-2072; Pessino et al. J. Exp. Med. 1998;188:953-960; Neri et al. Clin. Diag. Lab. Immun. 2001; 8:1131-1135;Pende et al. J. Exp. Med. 1999; 190:1505-1516, the entire disclosures ofeach of which are herein incorporated by reference.

In one embodiment, an antibody preparation causes at least a 10%augmentation in the cytotoxicity of a Siglec-restricted lymphocyte,preferably at least a 40% or 50% augmentation in NK cytotoxicity, ormore preferably at least a 70% augmentation in NK cytotoxicity.

The activity of a cytotoxic lymphocyte can also be addressed using acytokine-release assay, wherein NK cells are incubated with the antibodyto stimulate the cytokine production of the NK cells (for example IFN-γand TNF-α production). In an exemplary protocol, IFN-γ production fromPBMC is assessed by cell surface and intracytoplasmic staining andanalysis by flow cytometry after 4 days in culture. Briefly, Brefeldin A(Sigma Aldrich) is added at a final concentration of 5 μg/ml for thelast 4 hours of culture. The cells are then incubated with anti-CD3 andanti-CD56 mAb prior to permeabilization (IntraPrep™; Beckman Coulter)and staining with PE-anti-IFN-γ or PE-IgG1 (Pharmingen). GM-CSF andIFN-γ production from polyclonal activated NK cells are measured insupernatants using ELISA (GM-CSF: DuoSet Elisa, R&D Systems,Minneapolis, Minn., IFN-γ: OptEIA set, Pharmingen).

Fragments and derivatives of antibodies (which are encompassed by theterm “antibody” or “antibodies” as used in this application, unlessotherwise stated or clearly contradicted by context) can be produced bytechniques that are known in the art. “Fragments” comprise a portion ofthe intact antibody, generally the antigen binding site or variableregion. Examples of antibody fragments include Fab, Fab′, Fab′-SH, F(ab′) 2, and Fv fragments; diabodies; any antibody fragment that is apolypeptide having a primary structure consisting of one uninterruptedsequence of contiguous amino acid residues (referred to herein as a“single-chain antibody fragment” or “single chain polypeptide”),including without limitation (1) single-chain Fv molecules (2) singlechain polypeptides containing only one light chain variable domain, or afragment thereof that contains the three CDRs of the light chainvariable domain, without an associated heavy chain moiety and (3) singlechain polypeptides containing only one heavy chain variable region, or afragment thereof containing the three CDRs of the heavy chain variableregion, without an associated light chain moiety; and multispecific(e.g., bispecific) antibodies formed from antibody fragments. Included,inter alia, are a nanobody, domain antibody, single domain antibody or a“dAb”.

In certain embodiments, the DNA of a hybridoma producing an antibody,can be modified prior to insertion into an expression vector, forexample, by substituting the coding sequence for human heavy- andlight-chain constant domains in place of the homologous non-humansequences (e.g., Morrison et al., PNAS pp. 6851 (1984)), or bycovalently joining to the immunoglobulin coding sequence all or part ofthe coding sequence for a non-immunoglobulin polypeptide. In thatmanner, “chimeric” or “hybrid” antibodies are prepared that have thebinding specificity of the original antibody. Typically, suchnon-immunoglobulin polypeptides are substituted for the constant domainsof an antibody.

Optionally an antibody is humanized. “Humanized” forms of antibodies arespecific chimeric immunoglobulins, immunoglobulin chains or fragmentsthereof (such as Fv, Fab, Fab′, F (ab′) 2, or other antigen-bindingsubsequences of antibodies) which contain minimal sequence derived fromthe murine immunoglobulin. For the most part, humanized antibodies arehuman immunoglobulins (recipient antibody) in which residues from acomplementary-determining region (CDR) of the recipient are replaced byresidues from a CDR of the original antibody (donor antibody) whilemaintaining the desired specificity, affinity, and capacity of theoriginal antibody.

In some instances, Fv framework residues of the human immunoglobulin maybe replaced by corresponding non-human residues. Furthermore, humanizedantibodies can comprise residues that are not found in either therecipient antibody or in the imported CDR or framework sequences. Thesemodifications are made to further refine and optimize antibodyperformance. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the CDR regions correspond to thoseof the original antibody and all or substantially all of the FR regionsare those of a human immunoglobulin consensus sequence. The humanizedantibody optimally also will comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin. For further details see Jones et al., Nature, 321, pp.522 (1986); Reichmann et al, Nature, 332, pp. 323 (1988); Presta, Curr.Op. Struct. Biol., 2, pp. 593 (1992); Verhoeyen et Science, 239, pp.1534; and U.S. Pat. No. 4,816,567, the entire disclosures of which areherein incorporated by reference.) Methods for humanizing the antibodiesare well known in the art.

The choice of human variable domains, both light and heavy, to be usedin making the humanized antibodies is very important to reduceantigenicity. According to the so-called “best-fit” method, the sequenceof the variable domain of an antibody is screened against the entirelibrary of known human variable-domain sequences. The human sequencewhich is closest to that of the mouse is then accepted as the humanframework (FR) for the humanized antibody (Sims et al., J. Immunol. 151,pp. 2296 (1993); Chothia and Lesk, J. Mol. 196, 1987, pp. 901). Anothermethod uses a particular framework from the consensus sequence of allhuman antibodies of a particular subgroup of light or heavy chains. Thesame framework can be used for several different humanized antibodies(Carter et al., PNAS 89, pp. 4285 (1992); Presta et al., J. Immunol.,151, p. 2623 (1993)).

It is further important that antibodies be humanized with retention ofhigh affinity for Siglec receptors and other favorable biologicalproperties. To achieve this goal, according to one method, humanizedantibodies are prepared by a process of analysis of the parentalsequences and various conceptual humanized products usingthree-dimensional models of the parental and humanized sequences.Three-dimensional immunoglobulin models are commonly available and arefamiliar to those skilled in the art. Computer programs are availablewhich illustrate and display probable three-dimensional structures ofselected candidate immunoglobulin sequences. Inspection of thesedisplays permits analysis of the likely role of the residues in thefunctioning of the candidate immunoglobulin sequence, i.e., the analysisof residues that influence the ability of the candidate immunoglobulinto bind its antigen. In this way, FR residues can be selected andcombined from the consensus and import sequences so that the desiredantibody characteristic, such as increased affinity for the targetantigen (s), is achieved. In general, the CDR residues are directly andmost substantially involved in influencing antigen binding.

Another method of making “humanized” monoclonal antibodies is to use aXenoMouse (Abgenix, Fremont, Calif.) as the mouse used for immunization.A XenoMouse is a murine host according that has had its immunoglobulingenes replaced by functional human immunoglobulin genes. Thus,antibodies produced by this mouse or in hybridomas made from the B cellsof this mouse, are already humanized. The XenoMouse is described in U.S.Pat. No. 6,162,963, which is herein incorporated in its entirety byreference.

Human antibodies may also be produced according to various othertechniques, such as by using, for immunization, other transgenic animalsthat have been engineered to express a human antibody repertoire(Jakobovitz et al., Nature 362 (1993) 255), or by selection of antibodyrepertoires using phage display methods. Such techniques are known tothe skilled person and can be implemented starting from monoclonalantibodies as disclosed in the present application.

In one embodiment, the anti-Siglec antibodies can be prepared such thatthey do not have substantial specific binding to human FCγ receptors,e.g., any one or more of CD16A, CD16B, CD32A, CD32B and/or CD64). Suchantibodies may comprise constant regions of various heavy chains thatare known to lack or have low binding to FCγ receptors. Alternatively,antibody fragments that do not comprise (or comprise portions of)constant regions, such as F(ab′)2 fragments, can be used to avoid Fcreceptor binding. Fc receptor binding can be assessed according tomethods known in the art, including for example testing binding of anantibody to Fc receptor protein in a BIACORE assay. Also, generally anyantibody IgG isotype can be used in which the Fc portion is modified(e.g., by introducing 1, 2, 3, 4, 5 or more amino acid substitutions) tominimize or eliminate binding to Fc receptors (see, e.g., WO 03/101485,the disclosure of which is herein incorporated by reference). Assayssuch as cell based assays, to assess Fc receptor binding are well knownin the art, and are described in, e.g., WO 03/101485.

In one embodiment, the antibody can comprise one or more specificmutations in the Fc region that result in “Fc silent” antibodies thathave minimal interaction with effector cells. Silenced effectorfunctions can be obtained by mutation in the Fc region of the antibodiesand have been described in the art: N297A mutation, the LALA mutations,(Strohl, W., 2009, Curr. Opin. Biotechnol. vol. 20(6):685-691); andD265A (Baudino et al., 2008, J. Immunol. 181: 6664-69) see also Heusseret al., WO2012/065950, the disclosures of which are incorporated hereinby reference. In one embodiment, an antibody comprises one, two, threeor more amino acid substitutions in the hinge region. In one embodiment,the antibody is an IgG1 or IgG2 and comprises one, two or threesubstitutions at residues 233-236, optionally 233-238 (EU numbering). Inone embodiment, the antibody is an IgG4 and comprises one, two or threesubstitutions at residues 327, 330 and/or 331 (EU numbering). Examplesof silent Fc IgG1 antibodies are the LALA mutant comprising L234A andL235A mutation in the IgG1 Fc amino acid sequence. Another example of anFc silent mutation is a mutation at residue D265, or at D265 and P329for example as used in an IgG1 antibody as the DAPA (D265A, P329A)mutation (U.S. Pat. No. 6,737,056). Another silent IgG1 antibodycomprises a mutation at residue N297 (e.g. N297A, N297S mutation), whichresults in aglycosylated/non-glycosylated antibodies. Other silentmutations include: substitutions at residues L234 and G237(L234A/G237A); substitutions at residues S228, L235 and R409(S228P/L235E/R409K,T,M,L); substitutions at residues H268, V309, A330and A331 (H268Q/V309L/A330S/A331S); substitutions at residues C220,C226, C229 and P238 (02205/C226S/C229S/P238S); substitutions at residuesC226, C229, E233, L234 and L235 (C226S/C229S/E233P/L234V/L235A;substitutions at residues K322, L235 and L235 (K322A/L234A/L235A);substitutions at residues L234, L235 and P331 (L234F/L235E/P331S);substitutions at residues 234, 235 and 297; substitutions at residuesE318, K320 and K322 (L235E/E318A/K320A/K322A); substitutions at residues(V234A, G237A, P238S); substitutions at residues 243 and 264;substitutions at residues 297 and 299; substitutions such that residues233, 234, 235, 237, and 238 defined by the EU numbering system, comprisea sequence selected from PAAAP, PAAAS and SAAAS (see WO2011/066501).

In one embodiment, the antibody can comprise one or more specificmutations in the Fc region that result in improved stability of anantibody of the disclosure, e.g. comprising multiple aromatic amino acidresidues and/or having high hydrophobicity. For example, such anantibody can comprise an Fc domain of human IgG1 origin, comprises amutation at Kabat residue(s) 234, 235, 237, 330 and/or 331. One exampleof such an Fc domain comprises substitutions at Kabat residues L234,L235 and P331 (e.g., L234A/L235E/P331S or (L234F/L235E/P331S). Anotherexample of such an Fc domain comprises substitutions at Kabat residuesL234, L235, G237 and P331 (e.g., L234A/L235E/G237A/P331S). Anotherexample of such an Fc domain comprises substitutions at Kabat residuesL234, L235, G237, A330 and P331 (e.g., L234A/L235E/G237A/A330S/P331S).In one embodiment, the antibody comprises an Fc domain, optionally ofhuman IgG1 isotype, comprising: a L234X₁ substitution, a L235X₂substitution, and a P331X₃ substitution, wherein X₁ is any amino acidresidue other than leucine, X₂ is any amino acid residue other thanleucine, and X₃ is any amino acid residue other than proline; optionallywherein X₁ is an alanine or phenylalanine or a conservative substitutionthereof; optionally wherein X₂ is glutamic acid or a conservativesubstitution thereof; optionally wherein X₃ is a serine or aconservative substitution thereof. In another embodiment, the antibodycomprises an Fc domain, optionally of human IgG1 isotype, comprising: aL234X₁ substitution, a L235X₂ substitution, a G237X₄ substitution and aP331X₄ substitution, wherein X₁ is any amino acid residue other thanleucine, X₂ is any amino acid residue other than leucine, X₃ is anyamino acid residue other than glycine, and X₄ is any amino acid residueother than proline; optionally wherein X₁ is an alanine or phenylalanineor a conservative substitution thereof; optionally wherein X₂ isglutamic acid or a conservative substitution thereof; optionally, X₃ isalanine or a conservative substitution thereof; optionally X₄ is aserine or a conservative substitution thereof. In another embodiment,the antibody comprises an Fc domain, optionally of human IgG1 isotype,comprising: a L234X₁ substitution, a L235X₂ substitution, a G237X₄substitution, G330X₄ substitution, and a P331X₅ substitution, wherein X₁is any amino acid residue other than leucine, X₂ is any amino acidresidue other than leucine, X₃ is any amino acid residue other thanglycine, X₄ is any amino acid residue other than alanine, and X₅ is anyamino acid residue other than proline; optionally wherein X₁ is analanine or phenylalanine or a conservative substitution thereof;optionally wherein X₂ is glutamic acid or a conservative substitutionthereof; optionally, X₃ is alanine or a conservative substitutionthereof; optionally, X₄ is serine or a conservative substitutionthereof; optionally X₅ is a serine or a conservative substitutionthereof. In the shorthand notation used here, the format is: Wild typeresidue: Position in polypeptide: Mutant residue, wherein residuepositions are indicated according to EU numbering according to Kabat.

In one embodiment, an antibody comprises a heavy chain constant regioncomprising the amino acid sequence below, or an amino acid sequence atleast 90%, 95% or 99% identical thereto but retaining the amino acidresidues at Kabat positions 234, 235 and 331 (underlined):

(SEQ ID NO: 166) A S T K G P S V F P L A P S S K S T S G G T A A LG C L V K D Y F P E P V T V S W N S G A L T S G VH T F P A V L Q S S G L Y S L S S V V T V P S S SL G T Q T Y I C N V N H K P S N T K V D K R V E PK S C D K T H T C P P C P A P E  A   E  G G P S V F LF P P K P K D T L M I S R T P E V T C V V V D V SH E D P E V K F N W Y V D G V E V H N A K T K P RE E Q Y N S T Y R V V S V L T V L H Q D W L N G KE Y K C K V S N K A L P A  S  I E K T I S K A K G QP R E P Q V Y T L P P S R E E M T K N Q V S L T CL V K G F Y P S D I A V E W E S N G Q P E N N Y KT T P P V L D S D G S F F L Y S K L T V D K S R WQ Q G N V F S C S V M H E A L H N H Y T Q K S L S L S P G K

In one embodiment, an antibody comprises a heavy chain constant regioncomprising the amino acid sequence below, or an amino acid sequence atleast 90%, 95% or 99% identical thereto but retaining the amino acidresidues at Kabat positions 234, 235 and 331 (underlined):

(SEQ ID NO: 167) A S T K G P S V F P L A P S S K S T S G G T A A LG C L V K D Y F P E P V T V S W N S G A L T S G VH T F P A V L Q S S G L Y S L S S V V T V P S S SL G T Q T Y I C N V N H K P S N T K V D K R V E PK S C D K T H T C P P C P A P E  F   E  G G P S V F LF P P K P K D T L M I S R T P E V T C V V V D V SH E D P E V K F N W Y V D G V E V H N A K T K P RE E Q Y N S T Y R V V S V L T V L H Q D W L N G KE Y K C K V S N K A L P A  S  I E K T I S K A K G QP R E P Q V Y T L P P S R E E M T K N Q V S L T CL V K G F Y P S D I A V E W E S N G Q P E N N Y KT T P P V L D S D G S F F L Y S K L T V D K S R WQ Q G N V F S C S V M H E A L H N H Y T Q K S L S L S P G K

In one embodiment, an antibody comprises a heavy chain constant regioncomprising the amino acid sequence below, or an amino acid sequence atleast 90%, 95% or 99% identical thereto but retaining the amino acidresidues at Kabat positions 234, 235, 237, 330 and 331 (underlined):

(SEQ ID NO: 168) A S T K G P S V F P L A P S S K S T S G G T A A LG C L V K D Y F P E P V T V S W N S G A L T S G VH T F P A V L Q S S G L Y S L S S V V T V P S S SL G T Q T Y I C N V N H K P S N T K V D K R V E PK S C D K T H T C P P C P A P E  A   E  G  A  P S V F LF P P K P K D T L M I S R T P E V T C V V V D V SH E D P E V K F N W Y V D G V E V H N A K T K P RE E Q Y N S T Y R V V S V L T V L H Q D W L N G KE Y K C K V S N K A L P  S   S  I E K T I S K A K G QP R E P Q V Y T L P P S R E E M T K N Q V S L T CL V K G F Y P S D I A V E W E S N G Q P E N N Y KT T P P V L D S D G S F F L Y S K L T V D K S R WQ Q G N V F S C S V M H E A L H N H Y T Q K S L S L S P G K

In one embodiment, an antibody comprises a heavy chain constant regioncomprising the amino acid sequence below, or a sequence at least 90%,95% or 99% identical thereto but retaining the amino acid residues atKabat positions 234, 235, 237 and 331 (underlined):

(SEQ ID NO: 169) A S T K G P S V F P L A P S S K S T S G G T A A L GC L V K D Y F P E P V T V S W N S G A L T S G V H TF P A V L Q S S G L Y S L S S V V T V P S S S L G TQ T Y I C N V N H K P S N T K V D K R V E P K S C DK T H T C P P C P A P E  A   E  G  A  P S V F L F P P K PK D T L M I S R T P E V T C V V V D V S H E D P E VK F N W Y V D G V E V H N A K T K P R E E Q Y N S TY R V V S V L T V L H Q D W L N G K E Y K C K V S N K A L P A  S I E K T I S K A K G Q P R E P Q V Y T LP P S R E E M T K N Q V S L T C L V K G F Y P S D IA V E W E S N G Q P E N N Y K T T P P V L D S D G SF F L Y S K L T V D K S R W Q Q G N V F S C S V M HE A L H N H Y T Q K S L S L S P G K

Fc silent antibodies result in no or low ADCC activity, meaning that anFc silent antibody exhibits an ADCC activity that is below 50% specificcell lysis. Preferably an antibody substantially lacks ADCC activity,e.g., the Fc silent antibody exhibits an ADCC activity (specific celllysis) that is below 5% or below 1%. Fc silent antibodies can alsoresult in lack of FcγR-mediated cross-linking of Siglec-9 and/orSiglec-7 at the surface of a cell (e.g. an NK cell, a T cell, amonocyte, a dendritic cell, a macrophage).

In one embodiment, the antibody has a substitution in a heavy chainconstant region at any one, two, three, four, five or more of residuesselected from the group consisting of: 220, 226, 229, 233, 234, 235,236, 237, 238, 243, 264, 268, 297, 298, 299, 309, 310, 318, 320, 322,327, 330, 331 and 409 (numbering of residues in the heavy chain constantregion is according to EU numbering according to Kabat). In oneembodiment, the antibody comprises a substitution at residues 234, 235and 322. In one embodiment, the antibody has a substitution at residues234, 235 and 331. In one embodiment, the antibody has a substitution atresidues 234, 235, 237 and 331. In one embodiment, the antibody has asubstitution at residues 234, 235, 237, 330 and 331. In one embodiment,the Fc domain is of human IgG1 subtype. Amino acid residues areindicated according to EU numbering according to Kabat.

Antibody CDR Sequences

The amino acid sequence of the heavy and light chain variable regions ofantibodies mAb1, -2, -3, -4, -5, -6, -A, -B, -C, -D, -E and —F are shownin Table B. In a specific embodiment, provided is an antibody that bindsessentially the same epitope or determinant as monoclonal antibodiesmAb1, -2, -3, -4, -5, -6, -A, -B, -C, -D, -E or -F; optionally theantibody comprises the hypervariable region of antibody mAb1, -2, -3,-4, -5, -6, -A, —B, —C, -D, -E or -F. In any of the embodiments herein,antibody mAb1, -2, -3, -4, -5, -6, -A, —B, —C, -D, -E or -F can becharacterized by the amino acid sequences and/or nucleic acid sequencesencoding it. In one embodiment, the monoclonal antibody comprises the VHand/or VL, or the Fab or F(ab′)₂ portion of mAb1, -2, -3, -4, -5, -6,-A, -B, -C, -D, -E or -F. Also provided is a monoclonal antibody thatcomprises the heavy chain variable region of mAb1. According to oneembodiment, the monoclonal antibody comprises the three CDRs of theheavy chain variable region of mAb1, -2, -3, -4, -5, -6, -A, -B, -C, -D,-E or -F Also provided is a monoclonal antibody that further comprisesthe variable light chain variable region of the respective mAb1, -2, -3,-4, -5, -6, -A, -B, -C, -D, -E or -F, or one, two or three of the CDRsof the light chain variable region of the respective mAb1, -2, -3, -4,-5, -6, -A, —B, —C, -D, -E or -F. Optionally any one or more of saidlight or heavy chain CDRs may contain one, two, three, four or five ormore amino acid modifications (e.g., substitutions, insertions ordeletions). Optionally, provided is an antibody where any of the lightand/or heavy chain variable regions comprising part or all of an antigenbinding region of antibody mAb1 are fused to an immunoglobulin constantregion of the human IgG type, optionally a human constant region,optionally a human IgG1, IgG2, IgG3 or IgG4 isotype, optionally furthercomprising an amino acid substitution to reduce effector function(binding to human Fcγ receptors).

In another aspect, provided is an antibody comprising: a HCDR1 region ofmAb1 comprising an amino acid sequence as set forth in Table A-1, or asequence of at least 4, 5, 6, 7, 8, 9 or 10 contiguous amino acidsthereof, optionally wherein one or more of these amino acids may besubstituted by a different amino acid; a HCDR2 region of mAb1 comprisingan amino acid sequence as set forth in Table A-1, or a sequence of atleast 4, 5, 6, 7, 8, 9 or 10 contiguous amino acids thereof, optionallywherein one or more of these amino acids may be substituted by adifferent amino acid; a HCDR3 region of mAb1 comprising an amino acidsequence as set forth in Table A-1, or a sequence of at least 4, 5, 6,7, 8, 9 or 10 contiguous amino acids thereof, optionally wherein one ormore of these amino acids may be substituted by a different amino acid;a LCDR1 region of mAb1 comprising an amino acid sequence as set forth inTable A-1, or a sequence of at least 4, 5, 6, 7, 8, 9 or 10 contiguousamino acids thereof, optionally wherein one or more of these amino acidsmay be substituted by a different amino acid; a LCDR2 region of mAb1comprising an amino acid sequence as set forth in Table A-1, or asequence of at least 4, 5, 6, 7, 8, 9 or 10 contiguous amino acidsthereof, optionally wherein one or more of these amino acids may besubstituted by a different amino acid; a LCDR3 region of mAb1 comprisingan amino acid sequence as set forth in Table A-1, or a sequence of atleast 4, 5, 6, 7, 8, 9 or 10 contiguous amino acids thereof, optionallywherein one or more of these amino acids may be deleted or substitutedby a different amino acid.

TABLE A-1 CDR HCDR1 HCDR2 HCDR3 mAb definition SEQ ID Sequence SEQ IDSequence SEQ ID Sequence mAb1 Kabat 27 GGFAWN 30 YIGYGGSTSYNPSLNS 32GDYLFAY Chotia 28 GYSITGGF YGG 33 DYLFA IMGT 29 GYSITGGFA 31 IGYGGST 34ARGDYLFAY CDR LCDR1 LCDR2 LCDR3 mAb definition SEQ Sequence SEQ IDSequence SEQ ID Sequence mAb1 Kabat 35 KASQDVNTA 38 SASYRYT 39 QQHYSTPRTVA Chotia 36 SQDVNTA SAS 40 HYSTPR IMGT 37 QDVNTA SAS 39 QQHYSTPRT

In another aspect, the invention provides an antibody, wherein theantibody comprises: a HCDR1 region of mAb3 comprising an amino acidsequence as set forth in Table A-3, or a sequence of at least 4, 5, 6,7, 8, 9 or 10 contiguous amino acids thereof, optionally wherein one ormore of these amino acids may be substituted by a different amino acid;a HCDR2 region of mAb3 comprising an amino acid sequence as set forth inTable A-3, or a sequence of at least 4, 5, 6, 7, 8, 9 or 10 contiguousamino acids thereof, optionally wherein one or more of these amino acidsmay be substituted by a different amino acid; a HCDR3 region of mAb3comprising an amino acid sequence as set forth in Table A-3, or asequence of at least 4, 5, 6, 7, 8, 9 or 10 contiguous amino acidsthereof, optionally wherein one or more of these amino acids may besubstituted by a different amino acid; a LCDR1 region of mAb3 comprisingan amino acid sequence as set forth in Table A-3, or a sequence of atleast 4, 5, 6, 7, 8, 9 or 10 contiguous amino acids thereof, optionallywherein one or more of these amino acids may be substituted by adifferent amino acid; a LCDR2 region of mAb3 comprising an amino acidsequence as set forth in Table A-3, or a sequence of at least 4, 5, 6,7, 8, 9 or 10 contiguous amino acids thereof, optionally wherein one ormore of these amino acids may be substituted by a different amino acid;a LCDR3 region of mAb3 comprising an amino acid sequence as set forth inTable A-3, or a sequence of at least 4, 5, 6, 7, 8, 9 or 10 contiguousamino acids thereof, optionally wherein one or more of these amino acidsmay be deleted or substituted by a different amino acid.

TABLE A-3 CDR HCDR1 HCDR2 HCDR3 mAb definition SEQ ID Sequence SEQ IDSequence SEQ ID Sequence mAb3 Kabat 27 GGFAWN 30 YIGYGGSTSYNPSLNS 32GDYLFAY Chotia 28 GYSITGGF YGG 33 DYLFA IMGT 29 GYSITGGFA 31 IGYGGST 34ARGDYLFAY CDR LCDR1 LCDR2 LCDR3 mAb definition SEQ Sequence SEQ IDSequence SEQ Sequence mAb3 Kabat 41 RASGNIHNYLA 44 NAKTLAD 45 QHFWSTPRTChotia 42 SGNIHNY NAK 46 FWSTPR IMGT 43 GNIHNY NAK 45 QHFWSTPRT

In another aspect, the invention provides an antibody, wherein theantibody comprises: a HCDR1 region of mAb4 comprising an amino acidsequence as set forth in Table A-4, or a sequence of at least 4, 5, 6,7, 8, 9 or 10 contiguous amino acids thereof, optionally wherein one ormore of these amino acids may be substituted by a different amino acid;a HCDR2 region of mAb4 comprising an amino acid sequence as set forth inTable A-4, or a sequence of at least 4, 5, 6, 7, 8, 9 or 10 contiguousamino acids thereof, optionally wherein one or more of these amino acidsmay be substituted by a different amino acid; a HCDR3 region of mAb4comprising an amino acid sequence as set forth in Table A-4, or asequence of at least 4, 5, 6, 7, 8, 9 or 10 contiguous amino acidsthereof, optionally wherein one or more of these amino acids may besubstituted by a different amino acid; a LCDR1 region of mAb4 comprisingan amino acid sequence as set forth in Table A-4, or a sequence of atleast 4, 5, 6, 7, 8, 9 or 10 contiguous amino acids thereof, optionallywherein one or more of these amino acids may be substituted by adifferent amino acid; a LCDR2 region of mAb4 comprising an amino acidsequence as set forth in Table A-4, or a sequence of at least 4, 5, 6,7, 8, 9 or 10 contiguous amino acids thereof, optionally wherein one ormore of these amino acids may be substituted by a different amino acid;a LCDR3 region of mAb4 comprising an amino acid sequence as set forth inTable A-4, or a sequence of at least 4, 5, 6, 7, 8, 9 or 10 contiguousamino acids thereof, optionally wherein one or more of these amino acidsmay be deleted or substituted by a different amino acid.

TABLE A-4 CDR HCDR1 HCDR2 HCDR3 mAb definition SEQ ID Sequence SEQ IDSequence SEQ Sequence mAb4 Kabat 47 SYDMS 50 HIGSGGGNIYYPDTVKG 52LIFTTGFYGMDY Chotia 48 GFAFSSY SGGG 53 IFTTGFYGMD IMGT 49 GFAFSSYD 51IGSGGGNI 54 ARLIFTTGFYGMDY CDR LCDR1 LCDR2 LCDR3 mAb definition SEQSequence SEQ ID Sequence SEQ Sequence mAb4 Kabat 55 RASQDISSYLN 58YTSRLHS 59 QQGNALPWT Chotia 56 SQDISSY YTS 60 GNALPW IMGT 57 QDISSY YTS59 QQGNALPWT

In another aspect, the invention provides an antibody, wherein theantibody comprises: a HCDR1 region of mAb5 comprising an amino acidsequence as set forth in Table A-5, or a sequence of at least 4, 5, 6,7, 8, 9 or 10 contiguous amino acids thereof, optionally wherein one ormore of these amino acids may be substituted by a different amino acid;a HCDR2 region of mAb5 comprising an amino acid sequence as set forth inTable A-5, or a sequence of at least 4, 5, 6, 7, 8, 9 or 10 contiguousamino acids thereof, optionally wherein one or more of these amino acidsmay be substituted by a different amino acid; a HCDR3 region of mAb5comprising an amino acid sequence as set forth in Table A-5, or asequence of at least 4, 5, 6, 7, 8, 9 or 10 contiguous amino acidsthereof, optionally wherein one or more of these amino acids may besubstituted by a different amino acid; a LCDR1 region of mAb5 comprisingan amino acid sequence as set forth in Table A-5, or a sequence of atleast 4, 5, 6, 7, 8, 9 or 10 contiguous amino acids thereof, optionallywherein one or more of these amino acids may be substituted by adifferent amino acid; a LCDR2 region of mAb5 comprising an amino acidsequence as set forth in Table A-5, or a sequence of at least 4, 5, 6,7, 8, 9 or 10 contiguous amino acids thereof, optionally wherein one ormore of these amino acids may be substituted by a different amino acid;a LCDR3 region of mAb5 comprising an amino acid sequence as set forth inTable A-5, or a sequence of at least 4, 5, 6, 7, 8, 9 or 10 contiguousamino acids thereof, optionally wherein one or more of these amino acidsmay be deleted or substituted by a different amino acid.

TABLE A-5 CDR HCDR1 HCDR2 HCDR3 mAb definition SEQ ID Sequence SEQ IDSequence SEQ ID Sequence mAb5 Kabat 61 DYNMN 64 NIDPYYGATSYNQRFKG 66GDSLFAY Chotia 62 GYSFSDY PYYG 67 DSLFA IMGT 63 GYSFSDYN 65 IDPYYGAT 68ARGDSLFAY CDR LCDR1 LCDR2 LCDR3 mAb definition SEQ Sequence SEQ IDSequence SEQ ID Sequence mAb5 Kabat 69 KASQNVGTNVA 72 SASSRYS 73QQYITYPYT Chotia 70 SQNVGTN SAS 74 YITYPY IMGT 71 QNVGTN SAS 73QQYITYPYT

In another aspect, the invention provides an antibody, wherein theantibody comprises: a HCDR1 region of mAbA comprising an amino acidsequence as set forth in Table A-7, or a sequence of at least 4, 5, 6,7, 8, 9 or 10 contiguous amino acids thereof, optionally wherein one ormore of these amino acids may be substituted by a different amino acid;a HCDR2 region of mAbA comprising an amino acid sequence as set forth inTable A-7 or a sequence of at least 4, 5, 6, 7, 8, 9 or 10 contiguousamino acids thereof, optionally wherein one or more of these amino acidsmay be substituted by a different amino acid; a HCDR3 region of mAbAcomprising an amino acid sequence as set forth in Table A-7, or asequence of at least 4, 5, 6, 7, 8, 9 or 10 contiguous amino acidsthereof, optionally wherein one or more of these amino acids may besubstituted by a different amino acid; a LCDR1 region of mAbA comprisingan amino acid sequence as set forth in Table A-7, or a sequence of atleast 4, 5, 6, 7, 8, 9 or 10 contiguous amino acids thereof, optionallywherein one or more of these amino acids may be substituted by adifferent amino acid; a LCDR2 region of mAbA comprising an amino acidsequence as set forth in Table A-7, or a sequence of at least 4, 5, 6,7, 8, 9 or 10 contiguous amino acids thereof, optionally wherein one ormore of these amino acids may be substituted by a different amino acid;a LCDR3 region of mAbA comprising an amino acid sequence as set forth inTable A-7, or a sequence of at least 4, 5, 6, 7, 8, 9 or 10 contiguousamino acids thereof, optionally wherein one or more of these amino acidsmay be deleted or substituted by a different amino acid.

TABLE A-7 CDR HCDR1 HCDR2 HCDR3 mAb definition SEQ ID Sequence SEQ IDSequence SEQ ID Sequence mAbA Kabat 75 SYWMH 78 EINPSNGHTNYNEKFES 80GVESYDFDDALDY Chotia 76 YFTFTSY PSNG 81 VESYDFDDALD IMGT 77 YFTFTSYW 79INPSNGHT 82 ANGVESYDFDDALDY CDR LCDR1 LCDR2 LCDR3 mAb definition SEQSequence SEQ ID Sequence SEQ ID Sequence mAbA Kabat 83 RASQDINNY 58YTSRLHS 86 QQGNTLPFT LN Chotia 84 SQDINNY YTS 87 GNTLPF IMGT 85 QDINNYYTS 86 QQGNTLPFT

In another aspect, the invention provides an antibody, wherein theantibody comprises: a HCDR1 region of mAbB comprising an amino acidsequence as set forth in Table A-8, or a sequence of at least 4, 5, 6,7, 8, 9 or 10 contiguous amino acids thereof, optionally wherein one ormore of these amino acids may be substituted by a different amino acid;a HCDR2 region of mAbB comprising an amino acid sequence as set forth inTable A-8 or a sequence of at least 4, 5, 6, 7, 8, 9 or 10 contiguousamino acids thereof, optionally wherein one or more of these amino acidsmay be substituted by a different amino acid; a HCDR3 region of mAbBcomprising an amino acid sequence as set forth in Table A-8, or asequence of at least 4, 5, 6, 7, 8, 9 or 10 contiguous amino acidsthereof, optionally wherein one or more of these amino acids may besubstituted by a different amino acid; a LCDR1 region of mAbB comprisingan amino acid sequence as set forth in Table A-8, or a sequence of atleast 4, 5, 6, 7, 8, 9 or 10 contiguous amino acids thereof, optionallywherein one or more of these amino acids may be substituted by adifferent amino acid; a LCDR2 region of mAbB comprising an amino acidsequence as set forth in Table A-8, or a sequence of at least 4, 5, 6,7, 8, 9 or 10 contiguous amino acids thereof, optionally wherein one ormore of these amino acids may be substituted by a different amino acid;a LCDR3 region of mAbB comprising an amino acid sequence as set forth inTable A-8, or a sequence of at least 4, 5, 6, 7, 8, 9 or 10 contiguousamino acids thereof, optionally wherein one or more of these amino acidsmay be deleted or substituted by a different amino acid.

TABLE A-8 CDR HCDR1 HCDR2 HCDR3 mAb definition SEQ ID Sequence SEQ IDSequence SEQ ID Sequence mAbB Kabat 75 SYWMH 90 EINPSNGHTNYNEKFKT 92GVETYDFDDAMDY Chotia 88 VYTFTSY PSNG 93 VETYDFDDAMD IMGT 89 VYTFTSYW 91INPSNGHT 94 ANGVETYDFDDA MDY CDR LCDR1 LCDR2 LCDR3 mAb definition SEQSequence SEQ ID Sequence SEQ ID Sequence mAbB Kabat 83 RASQDINNYLN 95FTSRLHS 96 QQGDTFPFT Chotia 84 SQDINNY YTS 97 GDTFPF IMGT 85 QDINNY FTS96 QQGDTFPFT

In another aspect, the invention provides an antibody, wherein theantibody comprises: a HCDR1 region of mAbC comprising an amino acidsequence as set forth in Table A-9, or a sequence of at least 4, 5, 6,7, 8, 9 or 10 contiguous amino acids thereof, optionally wherein one ormore of these amino acids may be substituted by a different amino acid;a HCDR2 region of mAbC comprising an amino acid sequence as set forth inTable A-9 or a sequence of at least 4, 5, 6, 7, 8, 9 or 10 contiguousamino acids thereof, optionally wherein one or more of these amino acidsmay be substituted by a different amino acid; a HCDR3 region of mAbCcomprising an amino acid sequence as set forth in Table A-9, or asequence of at least 4, 5, 6, 7, 8, 9 or 10 contiguous amino acidsthereof, optionally wherein one or more of these amino acids may besubstituted by a different amino acid; a LCDR1 region of mAbC comprisingan amino acid sequence as set forth in Table A-9, or a sequence of atleast 4, 5, 6, 7, 8, 9 or 10 contiguous amino acids thereof, optionallywherein one or more of these amino acids may be substituted by adifferent amino acid; a LCDR2 region of mAbC comprising an amino acidsequence as set forth in Table A-9, or a sequence of at least 4, 5, 6,7, 8, 9 or 10 contiguous amino acids thereof, optionally wherein one ormore of these amino acids may be substituted by a different amino acid;a LCDR3 region of mAbC comprising an amino acid sequence as set forth inTable A-9, or a sequence of at least 4, 5, 6, 7, 8, 9 or 10 contiguousamino acids thereof, optionally wherein one or more of these amino acidsmay be deleted or substituted by a different amino acid.

TABLE A-9 CDR HCDR1 HCDR2 HCDR3 mAb definition SEQ ID Sequence SEQ IDSequence SEQ ID Sequence mAbC Kabat 98 NYEMN 101 WINTYTGESTYADDFK 103DDYGRSYGFAY Chotia 99 GYTFTNY TYTG 104 DYGRSYGFA IMGT 100 GYTFTNYE 102INTYTGES 105 VRDDYGRSYG FAY CDR LCDR1 LCDR2 LCDR3 mAb definition SEQSequence SEQ ID Sequence SEQ ID Sequence mAbC Kabat 106 RASESVDSYGN 109LASKLES 110 HQNNEDPPWT SFMH Chotia 107 SESVDSYGNSF LAS 111 NNEDPPW IMGT108 ESVDSYGNSF LAS 110 HQNNEDPPWT

In another aspect, the invention provides an antibody, wherein theantibody comprises: a HCDR1 region of mAbD comprising an amino acidsequence as set forth in Table A-10, or a sequence of at least 4, 5, 6,7, 8, 9 or 10 contiguous amino acids thereof, optionally wherein one ormore of these amino acids may be substituted by a different amino acid;a HCDR2 region of mAbD comprising an amino acid sequence as set forth inTable A-10 or a sequence of at least 4, 5, 6, 7, 8, 9 or 10 contiguousamino acids thereof, optionally wherein one or more of these amino acidsmay be substituted by a different amino acid; a HCDR3 region of mAbDcomprising an amino acid sequence as set forth in Table A-10, or asequence of at least 4, 5, 6, 7, 8, 9 or 10 contiguous amino acidsthereof, optionally wherein one or more of these amino acids may besubstituted by a different amino acid; a LCDR1 region of mAbD comprisingan amino acid sequence as set forth in Table A-10, or a sequence of atleast 4, 5, 6, 7, 8, 9 or 10 contiguous amino acids thereof, optionallywherein one or more of these amino acids may be substituted by adifferent amino acid; a LCDR2 region of mAbD comprising an amino acidsequence as set forth in Table A-10, or a sequence of at least 4, 5, 6,7, 8, 9 or 10 contiguous amino acids thereof, optionally wherein one ormore of these amino acids may be substituted by a different amino acid;a LCDR3 region of mAbD comprising an amino acid sequence as set forth inTable A-10, or a sequence of at least 4, 5, 6, 7, 8, 9 or 10 contiguousamino acids thereof, optionally wherein one or more of these amino acidsmay be deleted or substituted by a different amino acid.

TABLE A-10 CDR HCDR1 HCDR2 HCDR3 mAb definition SEQ ID Sequence SEQ IDSequence SEQ ID Sequence mAbD Kabat 112 DYSMH 115 WIITETGEPTYADDFRG 117DFDGY Chotia 113 GYTFTDY TETG FDG IMGT 114 GYTFTDYS 116 IITETGEP 118ARDFDGY CDR LCDR1 LCDR2 LCDR3 mAb definition SEQ Sequence SEQ IDSequence SEQ ID Sequence mAbD Kabat 119 RASENIYSYLA 122 NAKTLTE 123QHHYGFPWT Chotia 120 SENIYSY NAK 124 HYGFPW IMGT 121 ENIYSY NAK 123QHHYGFPWT

In another aspect, the invention provides an antibody, wherein theantibody comprises: a HCDR1 region of mAbE comprising an amino acidsequence as set forth in Table A-11, or a sequence of at least 4, 5, 6,7, 8, 9 or 10 contiguous amino acids thereof, optionally wherein one ormore of these amino acids may be substituted by a different amino acid;a HCDR2 region of mAbE comprising an amino acid sequence as set forth inTable A-11 or a sequence of at least 4, 5, 6, 7, 8, 9 or 10 contiguousamino acids thereof, optionally wherein one or more of these amino acidsmay be substituted by a different amino acid; a HCDR3 region of mAbEcomprising an amino acid sequence as set forth in Table A-11, or asequence of at least 4, 5, 6, 7, 8, 9 or 10 contiguous amino acidsthereof, optionally wherein one or more of these amino acids may besubstituted by a different amino acid; a LCDR1 region of mAbE comprisingan amino acid sequence as set forth in Table A-11, or a sequence of atleast 4, 5, 6, 7, 8, 9 or 10 contiguous amino acids thereof, optionallywherein one or more of these amino acids may be substituted by adifferent amino acid; a LCDR2 region of mAbE comprising an amino acidsequence as set forth in Table A-11, or a sequence of at least 4, 5, 6,7, 8, 9 or 10 contiguous amino acids thereof, optionally wherein one ormore of these amino acids may be substituted by a different amino acid;a LCDR3 region of mAbE comprising an amino acid sequence as set forth inTable A-11, or a sequence of at least 4, 5, 6, 7, 8, 9 or 10 contiguousamino acids thereof, optionally wherein one or more of these amino acidsmay be deleted or substituted by a different amino acid.

TABLE A-11 CDR HCDR1 HCDR2 HCDR3 mAb definition SEQ ID Sequence SEQ IDSequence SEQ ID Sequence mAbE Kabat 125 TFGMH 128 YISSGSNAIYYADTVKG 130PGYGAWFAY Chotia 126 GFTFSTF SGSN 131 GYGAWFA IMGT 127 GFTFSTFG 129ISSGSNAI 132 ASPGYGAWFAY CDR LCDR1 LCDR2 LCDR3 mAb definition SEQSequence SEQ ID Sequence SEQ ID Sequence mAbE Kabat 133 RASSSVSSAYLH 136STSNLAS 137 QQYSAYPYT Chotia 134 SSSVSSAY STS 138 YSAYPY IMGT 135SSVSSAY STS 137 QQYSAYPYT

In another aspect, the invention provides an antibody, wherein theantibody comprises: a HCDR1 region of mAbF comprising an amino acidsequence as set forth in Table A-12, or a sequence of at least 4, 5, 6,7, 8, 9 or 10 contiguous amino acids thereof, optionally wherein one ormore of these amino acids may be substituted by a different amino acid;a HCDR2 region of mAbF comprising an amino acid sequence as set forth inTable A-12 or a sequence of at least 4, 5, 6, 7, 8, 9 or 10 contiguousamino acids thereof, optionally wherein one or more of these amino acidsmay be substituted by a different amino acid; a HCDR3 region of mAbFcomprising an amino acid sequence as set forth in Table A-12, or asequence of at least 4, 5, 6, 7, 8, 9 or 10 contiguous amino acidsthereof, optionally wherein one or more of these amino acids may besubstituted by a different amino acid; a LCDR1 region of mAbF comprisingan amino acid sequence as set forth in Table A-12, or a sequence of atleast 4, 5, 6, 7, 8, 9 or 10 contiguous amino acids thereof, optionallywherein one or more of these amino acids may be substituted by adifferent amino acid; a LCDR2 region of mAbF comprising an amino acidsequence as set forth in Table A-12, or a sequence of at least 4, 5, 6,7, 8, 9 or 10 contiguous amino acids thereof, optionally wherein one ormore of these amino acids may be substituted by a different amino acid;a LCDR3 region of mAbF comprising an amino acid sequence as set forth inTable A-12, or a sequence of at least 4, 5, 6, 7, 8, 9 or 10 contiguousamino acids thereof, optionally wherein one or more of these amino acidsmay be deleted or substituted by a different amino acid.

TABLE A-12 CDR HCDR1 HCDR2 HCDR3 mAb definition SEQ ID Sequence SEQ IDSequence SEQ ID Sequence mAbF Kabat 112 DYSMH 139 VISTYNGNTNYNQKFKG 141RGYYGSSSWFGY Chotia 113 GYTFTDY TYNG 142 GYYGSSSWFG IMGT 114 GYTFTDYS140 ISTYNGNT 143 ARRGYYGSSSW FGY CDR LCDR1 LCDR2 LCDR3 mAb definitionSEQ Sequence SEQ ID Sequence SEQ ID Sequence mAbF Kabat 144 KASQNVGTDVA147 SASYRYS 148 QQYNSFPYT Chotia 145 SQNVGTD SAS 149 YNSFPY IMGT 146QNVGTD SAS 148 QQYNSFPYT

In another aspect of any of the embodiments herein, any of the HCDR1, 2,3 and LCDR1, 2, 3 sequences can optionally be specified as all (or each,independently) being those of the Kabat numbering system (as indicatedin Table A-1 to A-12 for each CDR), those of the Chotia numbering systemas indicated in Table A-1 to A-12 for each CDR), those of the IMGTnumbering system as indicated in Table A-1 to A-12 for each CDR), or anyother suitable numbering system.

In another aspect of any of the embodiments herein, any of the CDRs 1, 2and 3 of the heavy and light chains of mAbA, mAbB, mAbC, mAbD, mAbE,mAbF, mAb1, mAb2, mAb3, mAb4, mAb5 or mAb6 may be characterized by asequence of at least 4, 5, 6, 7, 8, 9 or 10 contiguous amino acidsthereof, and/or as having an amino acid sequence that shares at least50%, 60%, 70%, 80%, 85%, 90% or 95% sequence identity with theparticular CDR or set of CDRs listed in the corresponding SEQ ID NO.

In any of the antibodies of the invention, e.g., mAbA, mAbB, mAbC, mAbD,mAbE, mAbF, mAb1, mAb2, mAb3, mAb4, mAb5 or mAb6, the specified variableregion and CDR sequences may comprise sequence modifications, e.g., asubstitution (1, 2, 3, 4, 5, 6, 7, 8 or more sequence modifications). Inone embodiment, a CDRs 1, 2 and/or 3 of the heavy and light chainscomprises one, two, three or more amino acid substitutions, where theresidue substituted is a residue present in a sequence of human origin.In one embodiment the substitution is a conservative modification. Aconservative sequence modification refers to an amino acid modificationthat does not significantly affect or alter the binding characteristicsof the antibody containing the amino acid sequence. Such conservativemodifications include amino acid substitutions, additions and deletions.Modifications can be introduced into an antibody of the invention bystandard techniques known in the art, such as site-directed mutagenesisand PCR-mediated mutagenesis. Conservative amino acid substitutions aretypically those in which an amino acid residue is replaced with an aminoacid residue having a side chain with similar physicochemicalproperties. Specified variable region and CDR sequences may compriseone, two, three, four or more amino acid insertions, deletions orsubstitutions. Where substitutions are made, preferred substitutionswill be conservative modifications. Families of amino acid residueshaving similar side chains have been defined in the art. These familiesinclude amino acids with basic side chains (e.g., lysine, arginine,histidine), acidic side chains (e.g., aspartic acid, glutamic acid),uncharged polar side chains (e.g., glycine, asparagine, glutamine,serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains(e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine), beta-branched side chains (e.g., threonine, valine,isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine,tryptophan, histidine). Thus, one or more amino acid residues within theCDR regions of an antibody of the invention can be replaced with otheramino acid residues from the same side chain family and the alteredantibody can be tested for retained function (i.e., the properties setforth herein) using the assays described herein.

The sequences of the CDRs, according to IMGT, Kabat and Chothiadefinitions systems, are summarized in Tables A-1 to A-12. The sequencesof the variable regions of the antibodies according to the invention arelisted in Table B below. In any embodiment herein, a VL or VH sequencecan be specified or numbered so as to further comprise or lack a signalpeptide or any part thereof.

In one embodiment, the antibodies of the invention are antibodyfragments that retain their binding and/or functional properties.

TABLE B SEQ ID NO: Amino Acid Sequence mAb1 VH 3DVQLQESGPGLVKPSQSLSLTCTVTGYSITGGFAWNWIRQFPGNTLEWMGYIGYGGSTSYNPSLNSRISITRDTSKNHFFLQFNSVTTDDSATYYCARGDYLFAYWGQ GTLVTVSA mAb1 VL4 DIVMTQSHKFMSTSVGDRVSITCKASQDVNTAVAWYQQKPGQSPKLLIYSASYRYTGVPDRFTGSGSGTDFTFTISSVQAEDLAVYYCQQHYSTPRTFGGGTKLEIK mAb2 VH 5EVQLQESGPGLVKPSQSLSLTCTVTGYSITGGFAWNWIRQFPGNTLEWMGYIGYGGSTSYNPSLNSRISITRDTSKNHFFLQFNSVTTEDSATYYCARGDYLFAYWGQ GTLVTVSA mAb2 VL6 DIVMTQSHKFMSTSVGDRVSITCKASQDVNTAVAWYQQKPGQSPKLLIYSASYRYTGVPDRFTGSGSGTDFTFTISSVQAEDLAVYYCQQHYSTPRTFGGGTKLEIK mAb3 VH 7EVQLLETGPGLVKPSQSLSLTCTVTGYSITGGFAWNWIRQFPGNTLEWMGYIGYGGSTSYNPSLNSRISITRDTSKNHFFLQFNSVTTEDSATYYCARGDYLFAYWGQ GTLVTVSA mAb3 VL8 DILMTQSPASLSASVGETVSITCRASGNIHNYLAWYLQRQGKSPQLLVYNAKTLADGVPSRFSGTGSGTQFSLKINSLQPEDFGSYYCQHFWSTPRTFGGGTKLEIK mAb4 VH 9DVQLVESGGDLVKPGGSLKLSCAASGFAFSSYDMSWVRQSPEKRLEWIAHIGSGGGNIYYPDTVKGRFTISRDNAKNTLYLQMRSLKSEDTAMYYCARLIFTTGFYGM DYWGQGTSVTVSSmAb4 VL 10 DIQMTQTTSSLSASLGDRVTISCRASQDISSYLNWYQQKPDGTIKLLIYYTSRLHSGVPSRFSGSGSGTDYSLTISNLDQDDIATYFCQQGNALPWTFGGGTKLEIK mAb5 VH 11EIQLQQSGPELEKPGASVKISCKASGYSFSDYNMNWVKQSNGKSLEWIGNIDPYYGATSYNQRFKGKATLTVDKSSSTAYMQLKSLTSEDSAVYYCARGDSLFAYWGH GTLVTVSA mAb5 VL12 DIVMTQSQEFMSTSLGDRVSVTCKASQNVGTNVAWYQQKPGQSPKALLYSASSRYSGVPDRFTGSGSGTDFTLTISNVQSEDLAEYFCQQYITYPYTFGGGTKLEIK mAb6 VH 13EIQLQQSGPELEKPGASVKISCKASGYSFSDYNMNWVKQSNGKSLEWIGNIDPYYGATSYNQRFKGKATLTVDKSSSTAYMQLKSLTSEDSAVYYCARGDSLFAYWGQ GTLVTVSA mAb6 VL14 DIVMTQSQEFMSTSLGDRVSVTCKASQNVGTNVAWYQQKPGQSPKALLYSASSRYSGVPDRFTGSGSGTDFTLTINNMQSEDLAEYFCQQYITYPYTFGGGTKLEIK mAbA VH 15QVQLQQPGAELVKPGSPVKLSCKASYFTFTSYWMHWVRQRPGQGLEWIGEINPSNGHTNYNEKFESKATLTVDRSSSTAYMQLSSLTSEDSAVFYCANGVESYDFDDA LDYWGQGTSVTVSSmAbA VL 16 DIQMTQTTSSLSASLGDRVTISCRASQDINNYLNWYQQKPDGTIKLLIYYTSRLHSGVPSRFSGSGSGTDYSLTINNLEQEDIATYFCQQGNTLPFTFGGGTKLEIK mAbB VH 17QVQLQQPGAELVKPGASVKLSCKASVYTFTSYWMHWVKQRPGQGLEWIGEINPSNGHTNYNEKFKTKAKLTVDKSSSTAYMQLSSLTSEDSAVYFCANGVETYDFDDA MDYWGQGTSVTVSSmAbB VL 18 DIQMTQTTSSLSASLGDRVTISCRASQDINNYLNWYQQKPDGTVKLLIYFTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGDTFPFTFGGGTKLEIK mAbC VH 19QIQLVQSGPELKKPGETVKISCKASGYTFTNYEMNWVKEAPGKGLKWMGWINTYTGESTYADDFKGRFAFSLETSASTVYLQINNLKDEDVATYFCVRDDYGRSYGFA YWGQGTLVTVSAmAbC VL 20 NIVLTQSPASLTVSLGQRANISCRASESVDSYGNSFMHWYQQKPGQPPKLLIYLASKLESGVPARFSGSGSRTDFTLTIDPVETDDAATYYCHQNNEDPPWTFGGGTK LEIK mAbD VH 21QIQLVQSGPELKKPGETVKISCKASGYTFTDYSMHWVKQAPGKGLKWMGWIITETGEPTYADDFRGRFAFSLETSANTAYLQINNLKNEDTATYFCARDFDGYWGQGT TLTVSS mAbD VL 22DILMTQSPASLSASVGETVTITCRASENIYSYLAWYQQKRGKSPQFLVYNAKTLTEGVPSRFRGSGSGTQFSLKINSLQPEDFGTYYCQHHYGFPWTFGGGTKLEIK mAbE VH 23DVQLVESGGGLVQPGGSRKLSCAASGFTFSTFGMHWVRQAPEKGLEWVAYISSGSNAIYYADTVKGRFTISRDNPKNTLFLQMTSLRSEDTAMYYCASPGYGAWFAYW GQGTLVTVSAmAbE VL 24 ENVLTQSPAIMSASPGEKVTMTCRASSSVSSAYLHWYQQKSGASPKLWIYSTSNLASGVPARFSGSGSGTSYSLTISSVEAEDAATYYCQQYSAYPYTFGGGTKLEIK mAbF VH 25QVQLQQSGPEVVRPGVSVKISCKGSGYTFTDYSMHWVKQSHAKSLEWIGVISTYNGNTNYNQKFKGKATMTVDKSSSTAYMELARLTSEDSAIYYCARRGYYGSSSWF GYWGQGTLVTVSAmAbF VL 26 DIVMTQSQKFMSTSVGDRVSVTCKASQNVGTDVAWYQQKPGQSPEALIYSASYRYSGVPDRFTGSGSGADFTLTISNVQSEDLAEYFCQQYNSFPYTFGGGTKLEIK

Antibody Formulations

An anti-Siglec antibody can be incorporated in a pharmaceuticalformulation comprising in a concentration from 1 mg/ml to 500 mg/ml,wherein said formulation has a pH from 2.0 to 10.0. The formulation mayfurther comprise a buffer system, preservative(s), tonicity agent(s),chelating agent(s), stabilizers and surfactants. In one embodiment, thepharmaceutical formulation is an aqueous formulation, i.e., formulationcomprising water. Such formulation is typically a solution or asuspension. In a further embodiment, the pharmaceutical formulation isan aqueous solution. The term “aqueous formulation” is defined as aformulation comprising at least 50% w/w water. Likewise, the term“aqueous solution” is defined as a solution comprising at least 50% w/wwater, and the term “aqueous suspension” is defined as a suspensioncomprising at least 50% w/w water.

In another embodiment, the pharmaceutical formulation is a freeze-driedformulation, whereto the physician or the patient adds solvents and/ordiluents prior to use.

In another embodiment, the pharmaceutical formulation is a driedformulation (e.g., freeze-dried or spray-dried) ready for use withoutany prior dissolution.

In a further aspect, the pharmaceutical formulation comprises an aqueoussolution of such an antibody, and a buffer, wherein the antibody ispresent in a concentration from 1 mg/ml or above, and wherein saidformulation has a pH from about 2.0 to about 10.0.

In a another embodiment, the pH of the formulation is in the rangeselected from the list consisting of from about 2.0 to about 10.0, about3.0 to about 9.0, about 4.0 to about 8.5, about 5.0 to about 8.0, andabout 5.5 to about 7.5.

In a further embodiment, the buffer is selected from the groupconsisting of sodium acetate, sodium carbonate, citrate, glycylglycine,histidine, glycine, lysine, arginine, sodium dihydrogen phosphate,disodium hydrogen phosphate, sodium phosphate, andtris(hydroxymethyl)-aminomethan, bicine, tricine, malic acid, succinate,maleic acid, fumaric acid, tartaric acid, aspartic acid or mixturesthereof. Each one of these specific buffers constitutes an alternativeembodiment of the invention.

In a further embodiment, the formulation further comprises apharmaceutically acceptable preservative. In a further embodiment, theformulation further comprises an isotonic agent. In a furtherembodiment, the formulation also comprises a chelating agent. In afurther embodiment of the invention the formulation further comprises astabilizer. In a further embodiment, the formulation further comprises asurfactant. For convenience reference is made to Remington: The Scienceand Practice of Pharmacy, 19^(th) edition, 1995.

It is possible that other ingredients may be present in the peptidepharmaceutical formulation of the present invention. Such additionalingredients may include wetting agents, emulsifiers, antioxidants,bulking agents, tonicity modifiers, chelating agents, metal ions,oleaginous vehicles, proteins (e.g., human serum albumin, gelatine orproteins) and a zwitterion (e.g., an amino acid such as betaine,taurine, arginine, glycine, lysine and histidine). Such additionalingredients, of course, should not adversely affect the overallstability of the pharmaceutical formulation of the present invention.

Pharmaceutical compositions containing an antibody according to thepresent invention may be administered to a patient in need of suchtreatment at several sites, for example, at topical sites, for example,skin and mucosal sites, at sites which bypass absorption, for example,administration in an artery, in a vein, in the heart, and at sites whichinvolve absorption, for example, administration in the skin, under theskin, in a muscle or in the abdomen. Administration of pharmaceuticalcompositions according to the invention may be through several routes ofadministration, for example, subcutaneous, intramuscular,intraperitoneal, intravenous, lingual, sublingual, buccal, in the mouth,oral, in the stomach and intestine, nasal, pulmonary, for example,through the bronchioles and alveoli or a combination thereof, epidermal,dermal, transdermal, vaginal, rectal, ocular, for examples through theconjunctiva, uretal, and parenteral to patients in need of such atreatment.

Suitable antibody formulations can also be determined by examiningexperiences with other already developed therapeutic monoclonalantibodies. Several monoclonal antibodies have been shown to beefficient in clinical situations, such as Rituxan (Rituximab), Herceptin(Trastuzumab) Xolair (Omalizumab), Bexxar (Tositumomab), Campath(Alemtuzumab), Zevalin, Oncolym and similar formulations may be usedwith the antibodies of this invention. For example, a monoclonalantibody can be supplied at a concentration of 10 mg/mL in either 100 mg(10 mL) or 500 mg (50 mL) single-use vials, formulated for IVadministration in 9.0 mg/mL sodium chloride, 7.35 mg/mL sodium citratedihydrate, 0.7 mg/mL polysorbate 80, and Sterile Water for Injection.The pH is adjusted to 6.5. In another embodiment, the antibody issupplied in a formulation comprising about 20 mM Na-Citrate, about 150mM NaCl, at pH of about 6.0.

Diagnosis and Treatment of Malignancies

Methods of treating an individual, notably a human patient, using ananti-Siglec antibody as described herein are also provided for. In oneembodiment, the invention provides for the use of an antibody asdescribed herein in the preparation of a pharmaceutical composition foradministration to a human patient. Typically, the patient suffers from,or is at risk for, cancer or infections disease, e.g., a bacterial or aviral disease.

For example, in one aspect, the invention provides a method ofpotentiating the activity of Siglec-7 and/or -9-restricted immune cell,e.g., lymphocytes, in a patient in need thereof, comprising the step ofadministering a neutralizing anti-Siglec7 and/or -9 antibody to saidpatient. The antibody can be for example a human or humanizedanti-Siglec-7 and/or -9 antibody, which antibody reduces or preventssialic acid-mediated activation of the Siglec-7 and/or -9 receptors. Inone embodiment, the method directed at increasing the activity of suchlymphocytes in patients having a disease in which increased lymphocyte(e.g., NK and/or CD8+ T cell) activity is beneficial, which involves,affects or is caused by cells susceptible to lysis by NK or CD8+ Tcells, or which is caused or characterized by insufficient NK or CD8+ Tcell activity, such as a cancer or an infectious disease. For example,in one aspect, the invention provides a method of enhancing the activity(e.g. cellular activation, anti-tumor immunity or activity, cytokineproduction, proliferation) of Siglec-7 and/or -9-restricted immunecells, for example an NK cell (e.g. CD56^(bright) cell), a T cell, amonocyte, a dendritic cell, a macrophage (e.g., an immunosuppressive orM2 macrophage), in a patient in need thereof, comprising the step ofadministering a neutralizing anti-Siglec7 and/or -9 antibody of thedisclosure to said patient.

In one embodiment, the antibodies of the disclosure are used in thetreatment of a tumor characterized by expression of the ST3GAL6 and/orST3GAL1 enzyme (or, e.g., a high level of ST3GAL6 and/or ST3GAL1 enzymeactivity), optionally overexpression of the ST3GAL6 enzyme (compared toexpression in, e.g., healthy tissue, in healthy individuals).

More specifically, the methods and compositions herein are utilized forthe treatment of a variety of cancers and other proliferative diseases.Because these methods operate by enhancing an immune response viablockade of inhibitory receptors on lymphocytes, they are applicable toa very broad range of cancers. In one embodiment, a human patienttreated with an anti-Siglec antibody of the disclosure has liver cancer,bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck(e.g. HNSCC), breast cancer, lung cancer, non-small cell lung cancer(NSCLC), castrate resistant prostate cancer (CRPC), melanoma, uterinecancer, colon cancer, rectal cancer, cancer of the anal region, stomachcancer, testicular cancer, uterine cancer, carcinoma of the fallopiantubes, carcinoma of the endometrium, carcinoma of the cervix, carcinomaof the vagina, carcinoma of the vulva, non-Hodgkin's lymphoma, cancer ofthe esophagus, cancer of the small intestine, cancer of the endocrinesystem, cancer of the thyroid gland, cancer of the parathyroid gland,cancer of the adrenal gland, sarcoma of soft tissue, cancer of theurethra, cancer of the penis, solid tumors of childhood, lymphocyticlymphoma, cancer of the bladder, cancer of the kidney or ureter,carcinoma of the renal pelvis, neoplasm of the central nervous system(CNS), primary CNS lymphoma, tumor angiogenesis, spinal axis tumor,brain stem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoidcancer, squamous cell cancer, environmentally induced cancers includingthose induced by asbestos, hematologic malignancies including, forexample, multiple myeloma, B-cell lymphoma, Hodgkin lymphoma/primarymediastinal B-cell lymphoma, non-Hodgkin's lymphomas, acute myeloidlymphoma, chronic myelogenous leukemia, chronic lymphoid leukemia,follicular lymphoma, diffuse large B-cell lymphoma, Burkitt's lymphoma,immunoblastic large cell lymphoma, precursor B-lymphoblastic lymphoma,mantle cell lymphoma, acute lymphoblastic leukemia, mycosis fungoides,anaplastic large cell lymphoma, T-cell lymphoma, and precursorT-lymphoblastic lymphoma, and any combinations of said cancers. Thepresent invention is also applicable to treatment of metastatic cancers.Patients can be tested or selected for one or more of the abovedescribed clinical attributes prior to, during or after treatment.

The anti-Siglec antibody based treatment can also be used to treat orprevent infectious diseases, including preferably any infections causedby infection by viruses, bacteria, protozoa, molds or fungi. Such viralinfectious organisms include, but are not limited to, hepatitis type A,hepatitis type B, hepatitis type C, influenza, varicella, adenovirus,herpes simplex type I (HSV-1), herpes simplex type 2 (HSV-2),rinderpest, rhinovirus, echovirus, rotavirus, respiratory syncytialvirus, papilloma virus, papilloma virus, cytomegalovirus, echinovirus,arbovirus, huntavirus, coxsackie virus, mumps virus, measles virus,rubella virus, polio virus and human immunodeficiency virus type I ortype 2 (HIV-1, HIV-2). Bacteria constitute another preferred class ofinfectious organisms including but are not limited to the following:Staphylococcus; Streptococcus, including S. pyogenes; Enterococcl;Bacillus, including Bacillus anthracis, and Lactobacillus; Listeria;Corynebacterium diphtheriae; Gardnerella including G. vaginalis;Nocardia; Streptomyces; Thermoactinomyces vulgaris; Treponerna;Camplyobacter, Pseudomonas including P. aeruginosa; Legionella;Neisseria including N. gonorrhoeae and N. meningitides; Flavobacteriumincluding F. meningosepticum and F. odoraturn; Brucella; Bordetellaincluding B. pertussis and B. bronchiseptica; Escherichia including E.coli, Klebsiella; Enterobacter, Serratia including S. marcescens and S.liquefaciens; Edwardsiella; Proteus including P. mirabilis and P.vulgaris; Streptobacillus; Rickettsiaceae including R. fickettsfi,Chlamydia including C. psittaci and C. trachomatis; Mycobacteriumincluding M. tuberculosis, M. intracellulare, M. folluiturn, M. laprae,M. avium, M. bovis, M. africanum, M. kansasii, M. intracellulare, and M.lepraernurium; and Nocardia. Protozoa may include but are not limitedto, leishmania, kokzidioa, and trypanosoma. Parasites include but arenot limited to, Chlamydia and Rickettsia.

The antibody compositions may be used to treat individuals regardless ofthe residue present at position 100 in Siglec-9 (reference to SEQ ID NO:2) or position 104 in Siglec-7 (reference to SEQ ID NO: 1) in thealleles expressed by the individuals. Siglec-9 bearing a lysine atposition 100 (e.g. SEQ ID NO: 2) is representative of about 49% of thepopulation) while Siglec-9 bearing a glutamic acid at position 100 (e.g.SEQ ID NO: 160) is representative of about 36% of the population. In oneembodiment, the antibody compositions are used to treat individualshaving a lysine at position 100 in Siglec-9 (reference to SEQ ID NO: 2)and individuals having a glutamic acid at position 100 in Siglec-9. Inone embodiment, the same administration regimen is used to treatindividuals whose cells (e.g. NK cells, neutrophils, etc.) express alysine at position 100 in Siglec-9 (reference to SEQ ID NO: 2) andindividuals whose cells express a glutamic acid at position 100 inSiglec-9. In one embodiment, the administration regimen comprises thesame mode of administration, the same dosage and the same frequency ofadministration irrespective of the particular allele of MICA expressedin an individual.

The antibody compositions may be used in as monotherapy or combinedtreatments with one or more other therapeutic agents, including agentsnormally utilized for the particular therapeutic purpose for which theantibody is being administered. The additional therapeutic agent willnormally be administered in amounts and treatment regimens typicallyused for that agent in a monotherapy for the particular disease orcondition being treated. Such therapeutic agents include, but are notlimited to anti-cancer agents and chemotherapeutic agents.

In one embodiment, the anti-Siglec-9 and/or -7 neutralizing antibodieslack binding to human CD16 yet potentiate the activity ofCD16-expressing effector cells (e.g. NK or effector T cells).Accordingly, in one embodiment, the second or additional secondtherapeutic agent is an antibody or other Fc domain-containing proteincapable of inducing ADCC toward a cell to which it is bound, e.g. viaCD16 expressed by an NK cell. Typically, such antibody or other proteinwill comprise a domain that binds to an antigen of interest, e.g. anantigen present on a tumor cell (tumor antigen), and an Fc domain orportion thereof, and will exhibit binding to the antigen via the antigenbinding domain and to Fcγ receptors (e.g. CD16) via the Fc domain. Inone embodiment, its ADCC activity will be mediated at least in part byCD16. In one embodiment, the additional therapeutic agent is an antibodyhaving a native or modified human Fc domain, for example a Fc domainfrom a human IgG1 or IgG3 antibody. The term “antibody-dependentcell-mediated cytotoxicity” or “ADCC” is a term well understood in theart, and refers to a cell-mediated reaction in which non-specificcytotoxic cells that express Fc receptors (FcRs) recognize boundantibody on a target cell and subsequently cause lysis of the targetcell. Non-specific cytotoxic cells that mediate ADCC include naturalkiller (NK) cells, macrophages, monocytes, neutrophils, and eosinophils.The term “ADCC-inducing antibody” refers to an antibody thatdemonstrates ADCC as measured by assay(s) known to those of skill in theart. Such activity is typically characterized by the binding of the Fcregion with various FcRs. Without being limited by any particularmechanism, those of skill in the art will recognize that the ability ofan antibody to demonstrate ADCC can be, for example, by virtue of itsubclass (such as IgG1 or IgG3), by mutations introduced into the Fcregion, or by virtue of modifications to the carbohydrate patterns inthe Fc region of the antibody. Examples of antibodies that induce ADCCinclude rituximab (for the treatment of lymphomas, CLL, trastuzumab (forthe treatment of breast cancer), alemtuzumab (for the treatment ofchronic lymphocytic leukemia) and cetuximab (for the treatment ofcolorectal cancer, head and neck squamous cell carcinoma). Examples ofADCC-enhanced antibodies include but are not limited to: GA-101(hypofucosylated anti-CD20), margetuximab (Fc enhanced anti-HER2),mepolizumab, MEDI-551 (Fc engineered anti-CD19), obinutuzumab(glyco-engineered/hypofucosuylated anti-CD20), ocaratuzumab (Fcengineered anti-CD20), XmAb®5574/MOR208 (Fc engineered anti-CD19).

In one embodiment, the anti-Siglec-9 and/or -7 neutralizing antibodiesaugments the efficacy of agents that neutralizes the inhibitory activityof human PD-1, e.g. that inhibits the interaction between PD-1 andPD-L1, notably in individuals who are poor responders to (or notsensitive to) treatment with agent that neutralizes the inhibitoryactivity of human PD-1. The anti-Siglec-9 and/or -7 neutralizingantibodies may be useful to potentiate the activity of PD-1-expressingeffector cells (e.g. NK or effector T cells, e.g. Siglec-9 expressing NKcells). Accordingly, in one embodiment, the second or additional secondtherapeutic agent is an antibody or other agent that neutralizes theinhibitory activity of human PD-1.

Programmed Death 1 (PD-1) (also referred to as “Programmed Cell Death1”) is an inhibitory member of the CD28 family of receptors. Thecomplete human PD-1 sequence can be found under GenBank Accession No.U64863. Inhibition or neutralization the inhibitory activity of PD-1 caninvolve use of a polypeptide agent (e.g., an antibody, a polypeptidefused to an Fc domain, an immunoadhesin, etc.) that preventsPD-L1-induced PD-1 signalling. There are currently at least six agentsblocking the PD-1/PD-L1 pathway that are marketed or in clinicalevaluation. One agent is BMS-936558 (Nivolumab/ONO-4538, Bristol-MyersSquibb; formerly MDX-1106). Nivolumab, (Trade name Opdivo®) is anFDA-approved fully human IgG4 anti-PD-L1 mAb that inhibits the bindingof the PD-L1 ligand to both PD-1 and CD80 and is described as antibody5C4 in WO 2006/121168, the disclosure of which is incorporated herein byreference. For melanoma patients, the most significant OR was observedat a dose of 3 mg/kg, while for other cancer types it was at 10 mg/kg.Nivolumab is generally dosed at 10 mg/kg every 3 weeks until cancerprogression. The terms “reduces the inhibitory activity of human PD-1”,“neutralizes PD-1” or “neutralizes the inhibitory activity of humanPD-1” refers to a process in which PD-1 is inhibited in its signaltransduction capacity resulting from the interaction of PD-1 with one ormore of its binding partners, such as PD-L1 or PD-L2. An agent thatneutralizes the inhibitory activity of PD-1 decreases, blocks, inhibits,abrogates or interferes with signal transduction resulting from theinteraction of PD-1 with one or more of its binding partners, such asPD-L1, PD-L2. Such an agent can thereby reduce the negativeco-stimulatory signal mediated by or through cell surface proteinsexpressed on T lymphocytes, so as to enhance T-cell effector functionssuch as proliferation, cytokine production and/or cytotoxicity.

MK-3475 (human IgG4 anti-PD1 mAb from Merck), also referred to aslambrolizumab or pembrolizumab (Trade name Keytruda®) has been approvedby the FDA for the treatment of melanoma and is being tested in othercancers. Pembrolizumab was tested at 2 mg/kg or 10 mg/kg every 2 or 3weeks until disease progression. DNA constructs encoding the variableregions of the heavy and light chains of the humanized antibodies h409.All have been deposited with the American Type Culture Collection PatentDepository (10801 University Blvd., Manassas, Va.). The plasmidcontaining the DNA encoding the heavy chain of h409A-I 1 was depositedon Jun. 9, 2008 and identified as 081469_SPD-H and the plasmidcontaining the DNA encoding the light chain of h409AI 1 was deposited onJun. 9, 2008 and identified as 0801470_SPD-L-I 1. MK-3475, also known asMerck 3745 or SCH-900475, is also described in WO2009/114335.

MPDL3280A/RG7446 (anti-PD-L1 from Roche/Genentech) is a human anti-PD-L1mAb that contains an engineered Fc domain designed to optimize efficacyand safety by minimizing FcγR binding and consequentialantibody-dependent cellular cytotoxicity (ADCC). Doses of 10, 15, and 25mg/kg MPDL3280A were administered every 3 weeks for up to 1 year. Inphase 3 trial, MPDL3280A is administered at 1200 mg by intravenousinfusion every three weeks in NSCLC.

AMP-224 (Amplimmune and GSK) is an immunoadhesin comprising a PD-L2extracellular domain fused to an Fc domain. Other examples of agentsthat neutralize PD-1 may include an antibody that binds PD-L2 (ananti-PD-L2 antibody) and blocks the interaction between PD-1 and PD-L2.

Pidlizumab (CT-011; CureTech) (humanized IgG1 anti-PD1 mAb fromCureTech/Teva), Pidlizumab (CT-011; CureTech) (see e.g., WO2009/101611)is another example; the agent was tested in thirty patients withrituximab-sensitive relapsed FL were treated with 3 mg/kg intravenousCT-011 every 4 weeks for 4 infusions in combination with rituximab dosedat 375 mg/m2 weekly for 4 weeks, starting 2 weeks after the firstinfusion of CT-011.

Further known PD-1 antibodies and other PD-1 inhibitors include AMP-224(a B7-DC/IgG1 fusion protein licensed to GSK), AMP-514 described in WO2012/145493, antibody MEDI-4736 (an anti-PD-L1 developed byAstraZeneca/Medimmune) described in WO2011/066389 and US2013/034559,antibody YW243.55.S70 (an anti-PD-L1) described in WO2010/077634,MDX-1105, also known as BMS-936559, is an anti-PD-L1 antibody developedby Bristol-Myers Squibb described in WO2007/005874, and antibodies andinhibitors described in WO2006/121168, WO2009/014708, WO2009/114335 andWO2013/019906, the disclosures of which are hereby incorporated byreference. Further examples of anti-PD1 antibodies are disclosed inWO2015/085847 (Shanghai Hengrui Pharmaceutical Co. Ltd.), for exampleantibodies having light chain variable domain CDR1, 2 and 3 of SEQ IDNO: 6, SEQ ID NO: 7 and/or SEQ ID NO: 8, respectively, and antibodyheavy chain variable domain CDR1, 2 and 3 of SEQ ID NO: 3, SEQ ID NO: 4or SEQ ID NO: 5, respectively, wherein the SEQ ID NO references are thenumbering according to WO2015/085847, the disclosure of which isincorporated herein by reference. Antibodies that compete with any ofthese antibodies for binding to PD-1 or PD-L1 also can be used.

An exemplary anti-PD-1 antibody is pembrolizumab (see, e.g., WO2009/114335 the disclosure of which is incorporated herein byreference.). The anti-PD-1 antibody may be the antibody h409AI 1 in WO2008/156712, comprising heavy chain variable regions encoded by the DNAdeposited at the ATCC as 081469_SPD-H and light chain variable regionsencoded by the DNA deposited at the ATCC as0801470_SPD-L-I 1. In otherembodiments, the antibody comprises the heavy and light chain CDRs orvariable regions of pembrolizumab. Accordingly, in one embodiment, theantibody comprises the CDR1, CDR2, and CDR3 domains of the VH ofpembrolizumab encoded by the DNA deposited at the ATCC as 081469_SPD-H,and the CDR1, CDR2 and CDR3 domains of the VL of pembrolizumab encodedby the DNA deposited at the ATCC as 0801470_SPD-L-I 1.

In some embodiments, the PD-1 neutralizing agent is an anti-PD-L1 mAbthat inhibits the binding of PD-L1 to PD-1. In some embodiments, thePD-1 neutralizing agent is an anti-PD1 mAb that inhibits the binding ofPD-1 to PD-L1. In some embodiments, the PD-1 neutralizing agent is animmunoadhesin (e.g., an immunoadhesin comprising an extracellular orPD-1 binding portion of PD-L1 or PD-L2 fused to a constant region (e.g.,an Fc region of an immunoglobulin sequence).

In the treatment methods, the anti-Siglec antibody and the secondtherapeutic agent can be administered separately, together orsequentially, or in a cocktail. In some embodiments, the antigen-bindingcompound is administered prior to the administration of the secondtherapeutic agent. For example, the anti-Siglec antibody can beadministered approximately 0 to 30 days prior to the administration ofthe second therapeutic agent. In some embodiments, a Siglec-bindingcompound is administered from about 30 minutes to about 2 weeks, fromabout 30 minutes to about 1 week, from about 1 hour to about 2 hours,from about 2 hours to about 4 hours, from about 4 hours to about 6hours, from about 6 hours to about 8 hours, from about 8 hours to 1 day,or from about 1 to 5 days prior to the administration of the secondtherapeutic agent. In some embodiments, an anti-Siglec antibody isadministered concurrently with the administration of the therapeuticagents. In some embodiments, an anti-Siglec antibody is administeredafter the administration of the second therapeutic agent. For example,an anti-Siglec antibody can be administered approximately 0 to 30 daysafter the administration of the second therapeutic agent. In someembodiments, an anti-Siglec antibody is administered from about 30minutes to about 2 weeks, from about 30 minutes to about 1 week, fromabout 1 hour to about 2 hours, from about 2 hours to about 4 hours, fromabout 4 hours to about 6 hours, from about 6 hours to about 8 hours,from about 8 hours to 1 day, or from about 1 to 5 days after theadministration of the second therapeutic agent.

In other aspects, methods are provided for identifying Siglec-7+ and/orSiglec-9+NK cells and/or T cells, e.g., CD8 T cells, CD56^(bright) NKcells, CD56^(dim) NK cells. Assessing the co-expression of Siglec-7and/or Siglec-9 on NK cells and/or T cells can be used in diagnostic orprognostic methods. For example, a biological sample can be obtainedfrom an individual (e.g., from a blood sample, from cancer orcancer-adjacent tissue obtained from a cancer patient) and analyzed forthe presence of Siglec-7 and/or Siglec-9+NK and/or T cells. Theexpression of Siglec-9 on such cells can, for example, be used toidentify individuals having NK and/or T cells, for example tumorinfiltrating NK and/or T cells which are inhibited by Siglec-9polypeptides. The expression of both Siglec-7 and Siglec-9 on such cellscan, for example, be used to identify individuals having NK and/or Tcells, for example tumor infiltrating NK and/or T cells which areinhibited by both Siglec-7 and Siglec-9 polypeptides. The method can,for example, be useful as a prognostic for response to treatment with anagent that neutralizes Siglec-7 and/or Siglec-9. Expression of Siglec-9(and optionally further Siglec-7) on such cells can indicate anindividual suitable for treatment with an antibody of the disclosure.

In certain optional aspects, patients can be identified for treatmentwith an anti-Siglec-7 and/or -9 antibody by assessing the presence in atumor sample (e.g., tumor tissue and/or tumor adjacent tissue) ofnatural ligands for Siglec-7 and/or Siglec-9. In one embodiment of anyof the therapeutic uses or cancer treatment or prevention methodsherein, the treatment or prevention of a cancer in an individualcomprises:

a) determining whether malignant cells (e.g., tumor cells) within theindividual having a cancer express ligands of Siglec-7 and/or ligands ofSiglec-9, and

b) upon a determination that ligands of Siglec-7 and/or ligands ofSiglec-9 are significantly expressed by (e.g., on the surface of)malignant cells (e.g., tumor cells), administering to the individual arespective anti-Siglec7 and/or -9 antibody, e.g., an antibody accordingto any aspect of the disclosure.

In one embodiment, a determination that a biological sample (e.g., asample comprising tumor cells, tumor tissue and/or tumor adjacenttissue) prominently expresses ligands of Siglec-7 and/or ligands ofSiglec-9 indicates that the individual has a cancer that can be treatedwith and/or may receive benefit from an antibody that inhibits,respectively, a Siglec-7 and/or a Siglec-9 polypeptide.

In one embodiment, significant expression of ligands of Siglec-7 and/orligands of Siglec-9 means that said ligand(s) are expressed in asubstantial number of tumor cells taken from a given individual. Whilenot bound by a precise percentage value, in some examples a ligand canbe said to be “significantly expressed” if be present on at least 30%,40%, 50° %, 60%, 70%, 80%, or more of the tumor cells taken from apatient (in a sample).

In one embodiment of any of the methods, determining whether malignantcells (e.g., tumor cells) within the individual having a cancer expressligands of Siglec-7 and/or Siglec-9 comprises determining the level ofexpression of ligands of Siglec-7 and/or ligands of Siglec-9 onmalignant cells in a biological sample and comparing the level to areference level (e.g., a value, weak or strong cell surface staining,etc.). The reference level may, for example, correspond to a healthyindividual, to an individual deriving no/low clinical benefit fromtreatment with an anti-Siglec antibody, or to an individual derivingsubstantial clinical benefit from treatment with an anti-Siglecantibody. A determination that a biological sample expresses ligands ofSiglec-7 and/or ligands of Siglec-9 at a level that is increased (e.g.,a high value, strong surface staining, a level that corresponds to thatof an individual deriving substantial clinical benefit from treatmentwith an anti-Siglec antibody, a level that is higher than thatcorresponding to an individual deriving no/low clinical benefit fromtreatment with an anti-Siglec antibody, etc.) indicates that theindividual has a cancer that can be treated with an anti-Siglecantibody.

EXAMPLES Example 1: A Human NK Cell Subset that Co-Expresses BothSiglec-7 and Siglec-9

Among the CD33-related Siglecs, Siglec-7 (CD328) and Siglec-9 (CD329)share the property of binding to sialic acids, including glycansoverexpressed by cancer cells, and are thought to function as inhibitoryreceptors in the immune cells in which they are expressed. Toinvestigate the expression of Siglecs on lymphocytes, distribution ofSiglec-7 and Siglec-9 were studied on human NK cells.

Siglec-7 and Siglec-9 expression on NK cells was determined by flowcytometry on fresh NK cells purified from human donors. The NKpopulation was determined as CD3-CD56+ cells (anti CD3 Pacific blue—BDPharmingen #558124; anti CD56-PE-Vio770—Milteny #130 100 676).Anti-Siglec-7 antibody (clone 194211—IgG1 APC—R&D Systems #FAB11381A),anti-Siglec-9 antibody (clone 191240—IgG2A PE—R&D Systems #FAB1139P) andisotype controls IgG1 APC and IgG2A APC were used. NK cells wereincubated 30 min with 50 ul of staining Ab mix, washed twice withstaining buffer, and fluorescence was revealed with Canto II (HTS).

Results are shown in FIG. 1. A representative result is shown. MFI:Meanof fluorescence intensity. A significant fraction (about 44%) of NKcells expressed both Siglec-7 and Siglec-9, suggesting that a largeproportion of NK cells can be inhibited by each of (or both of) thesereceptors, as a function of the glycan ligands present, for example ontumor cells.

Example 2: Generation of Anti-Siglec Antibodies

To obtain anti-human Siglec-7 and Siglec-9 antibodies, Balb/c mice wereimmunized with a human Siglec-7 Fc and human Siglec-9 Fc extracellulardomain recombinant protein. Two different immunizations were done.

In a first immunization with Siglec-7 Fc and Siglec-9 Fc proteins, micereceived 2 injections of an emulsion of 30 μg of each protein andComplete Freund Adjuvant, intraperitoneally. Then, mice received a boostwith 7.5 μg of each protein, intravenously. Two different fusions(fusion 1 and 2) were done. Immune spleen cells were fused 3 days afterthe boost with X63.Ag8.653 immortalized B cells, and cultured in thepresence of irradiated spleen cells. Hybridomas were plated insemi-solid methylcellulose-containing medium and growing clones werepicked using a clonepix 2 apparatus (Molecular Devices). A secondimmunization was carried out, again with Siglec-7 Fc and Siglec-9 Fcproteins. Mice received 3 injections of an emulsion of 30 μg of eachprotein and Complete Freund Adjuvant, intraperitoneally. Then, micereceived a boost with 5 μg of each protein, intravenously. Threedifferent fusions (fusion 3, 4 and 5) were done. Immune spleen cellswere fused 3 days after the boost with X63.Ag8.653 immortalized B cells,and cultured in the presence of irradiated spleen cells. Hybridomas wereplated in medium in P96. Siglec-7 Fc and Siglec-9 Fc proteins used inthis immunization (and in the Examples hereafter) were produced in CHOcells. The Siglec-7 Fc protein had the following amino acid sequence:

(SEQ ID NO: 164) QKSNRKDYSLTMQSSVTVQEGMCVHVRCSFSYPVDSQTDSDPVHGYWFRAGNDISWKAPVATNNPAWAVQEETRDRFHLLGDPQTKNCTLSIRDARMSDAGRYFFRMEKGNIKWNYKYDQLSVNVTALTHRPNILIPGTLESGCFQNLTCSVPWACEQGTPPMISWMGTSVSPLHPSTTRSSVLTLIPQPQHHGTSLTCQVTLPGAGVTTNRTIQLNVSYPPQNLTVTVFQGEGTASTALGNSSSLSVLEGQSLRLVCAVDSNPPARLSWTWRSLTLYPSQPSNPLVLELQVHLGDEGEFTCRAQNSLGSQHVSLNLSLQQEYTGKMRPVSGVLLGAVGGGGSSPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSPGK.

The Siglec-9 Fc protein had the following amino acid sequence:

(SEQ ID NO: 165) QTSKLLTMQSSVTVQEGLCVHVPCSFSYPSHGWIYPGPVVHGYWFREGANTDQDAPVATNNPARAVWEETRDRFHLLGDPHTKNCTLSIRDARRSDAGRYFFRMEKGSIKWNYKHHRLSVNVTALTHRPNILIPGTLESGCPQNLTCSVPWACEQGTPPMISWIGTSVSPLDPSTTRSSVLTLIPQPQDHGTSLTCQVTFPGASVTTNKTVHLNVSYPPQNLTMTVFQGDGTVSTVLGNGSSLSLPEGQSLRLVCAVDAVDSNPPARLSLSWRGLTLCPSQPSNPGVLELPWVHLRDAAEFTCRAQNPLGSQQVYLNVSLQSKATSGVTQGGGGSSPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL SLSPGK

Primary screen: Supernatant (SN) of growing clones of both immunizationswere tested in a primary screen by flow cytometry using parental andhuSiglec-7, huSiglec-9 and cynoSiglec-expressing CHO cell lines.HuSiglec-7,—and cynoSiglec-expressing CHO were stained with 0.5 μM and0.05 μM CFSE, respectively. For the flow cytometry screening, all cellswere equally mixed and the presence of reacting antibodies insupernanants was revealed by Goat anti-mouse polyclonal antibody (pAb)labeled with alexa fluor 647.

Results: 20, 19 and more than 80 antibodies were selected in therespective fusions that bind to human Siglec-7 and/or Siglec-9 and/orSiglec-cyno in fusion 1, 2 and 3/4/5, respectively. Different crossreactive anti-Siglec-7, Siglec-9 and Siglec-Cyno antibodies andanti-Siglec-9 antibodies (that did not bind Siglec-7) from the 3different fusions were cloned and produced as chimeric human IgG1antibodies with a heavy chain N297Q (Kabat EU numbering) mutation whichresults in lack of N-linked glycosylation and diminished binding to Fcγreceptors.

FIG. 2 shows representative results from flow cytometry for examples ofantibodies that bind to Siglec-7 but not Siglec-9 or cynomolgus Siglec(right panel), that bind to each of Siglec-7, Siglec-9 and cynomolgusSiglec (middle panel), and that bind to Siglec-9 but not Siglec-7 orcynomolgus Siglec (left panel).

TABLE 1 Siglec sequences NCBI Reference Name Sequence Sequence (AA)Human NM_014385.3;QKSNRKDYSLTMQSSVTVQEGMCVHVRCSFSYPVDSQTDSDPVHGYWFRAGNDIS Siglec-7NP_055200.1 WKAPVATNNPAWAVQEETRDRFHLLGDPQTKNCTLSIRDARMSDAGRYFFRMEKGNIKWNYKYDQLSVNVTALTHRPNILIPGTLESGCFQNLTCSVPWACEQGTPPMISWMGTSVSPLHPSTTRSSVLTLIPQPQHHGTSLTCQVTLPGAGVTTNRTIQLNVSYPPQNLTVTVFQGEGTASTALGNSSSLSVLEGQSLRLVCAVDSNPPARLSWTWRSLTLYPSQPSNPLVLELQVHLGDEGEFTCRAQNSLGSQHVSLNLSLQQEYTGKMRPVSGVLLGAVGGAGATALVFLSFCVIFIVVRSCRKKSARPAADVGDIGMKDANTIRGSASQGNLTESWADDNPRHHGLAAHSSGEEREIQYAPLSFHKGEPQDLSGQEATNNEYSEIKIPK (SEQ ID NO: 150)Human NM_014441.1;QTSKLLTMQSSVTVQEGLCVHVPCSFSYPSHGWIYPGPVVHGYWFREGANTDQDAP Siglec-9NP_055256.1 VATNNPARAVWEETRDRFHLLGDPHTKNCTLSIRDARRSDAGRYFFRMEKGSIKWNYKHHRLSVNVTALTHRPNILIPGTLESGCPQNLTCSVPWACEQGTPPMISWIGTSVSPLDPSTTRSSVLTLIPQPQDHGTSLTCQVTFPGASVTTNKTVHLNVSYPPQNLTMTVFQGDGTVSTVLGNGSSLSLPEGQSLRLVCAVDAVDSNPPARLSLSWRGLTLCPSQPSNPGVLELPWVHLRDAAEFTCRAQNPLGSQQVYLNVSLQSKATSGVTQGVVGGAGATALVFLSFCVIFVVVRSCRKKSARPAAGVGDTGIEDANAVRGSASQGPLTEPWAEDSPPDQPPPASARSSVGEGELQYASLSFQMVKPWDSRGQEATDTEYSEIKIHR (SEQ ID NO: 151) HumanNM_001772.3; DPNFWLQVQESVTVQEGLCVLVPCTFFHPIPYYDKNSPVHGYWFREGAIISGDSPVATSiglec-3 NP_001763.3NKLDQEVQEETQGRFRLLGDPSRNNCSLSIVDARRRDNGSYFFRMERGSTKYSYKSPQLSVHVTDLTHRPKILIPGTLEPGHSKNLTCSVSWACEQGTPPIFSWLSAAPTSLGPRTTHSSVLIITPRPQDHGTNLTCQVKFAGAGVTTERTIQLNVTYVPQNPTTGIFPGDGSGKQETRAGVVHGAIGGAGVTALLALCLCLIFFIVKTHRRKAARTAVGRNDTHPTTGSASPKHQKKSKLHGPTETSSCSGAAPTVEMDEELHYASLNFHGMNPSKDTSTEYSEVRTQ (SEQ ID NO: 152) Human NM_003830.3EKPVYELQVQKSVTVQEGLCVLVPCSFSYPWRSWYSSPPLYVYWFRDGEIPYYAEVVA Siglec-5TNNPDRRVKPETQGRFRLLGDVQKKNCSLSIGDARMEDTGSYFFRVERGRDVKYSYQQNKLNLEVTALIEKPDIHFLEPLESGRPTRLSCSLPGSCEAGPPLTFSWTGNALSPLDPETTRSSELTLTPRPEDHGTNLTCQMKRQGAQVTTERTVQLNVSYAPQTITIFRNGIALEILQNTSYLPVLEGQALRLLCDAPSNPPAHLSWFQGSPALNATPISNTGILELRRVRSAEEGGFTCRAQHPLGFLQIFLNLSVYSLPQLLGPSCSWEAEGLHCRCSFRARPAPSLCWRLEEKPLEGNSSQGSFKVNSSSAGPWANSSLILHGGLSSDLKVSCKAWNIYGSQSGSVLLLQGRSNLGTGVVPAALGGAGVMALLCICLCLIFFLIVKARRKQAAGRPEKMDDEDPIMGTITSGSRKKPWPDSPGDQASPPGDAPPLEEQKELHYASLSFSEMKSREPKDQEAPSTTEYSEIKTSK (SEQ ID NO: 153) Human NM_198845.4QERRFQLEGPESLTVQEGLCVLVPCRLPTTLPASYYGYGYWFLEGADVPVATNDPDEE Siglec-6VQEETRGRFHLLWDPRRKNCSLSIRDARRRDNAAYFFRLKSKWMKYGYTSSKLSVRVMALTHRPNISIPGTLESGHPSNLTCSVPWVCEQGTPPIFSWMSAAPTSLGPRTTQSSVLTITPRPQDHSTNLTCQVTFPGAGVTMERTIQLNVSSFKILQNTSSLPVLEGQALRLLCDADGNPPAHLSWFQGFPALNATPISNTGVLELPQVGSAEEGDFTCRAQHPLGSLQISLSLFVHWKPEGRAGGVLGAVWGASITTLVFLCVCFIFRVKTRRKKAAQPVQNTDDVNPVMVSGSRGHQHQFQTGIVSDHPAEAGPISEDEQELHYAVLHFHKVQPQEPKVTDTEYSEIKIHK (SEQ ID NO: 154) Human NM_014442.2MEGDRQYGDGYLLQVQELVTVQEGLCVHVPCSFSYPQDGWTDSDPVHGYWFRAG Siglec-8DRPYQDAPVATNNPDREVQAETQGRFQLLGDIWSNDCSLSIRDARKRDKGSYFFRLERGSMKWSYKSQLNYKTKQLSVFVTALTHRPDILILGTLESGHSRNLTCSVPWACKQGTPPMISWIGASVSSPGPTTARSSVLTLTPKPQDHGTSLTCQVTLPGTGVTTTSTVRLDVSYPPWNLTMTVFQGDATASTALGNGSSLSVLEGQSLRLVCAVNSNPPARLSWTRGSLTLCPSRSSNPGLLELPRVHVRDEGEFTCRAQNAQGSQHISLSLSLQNEGTGTSRPVSQVTLAAVGGAGATALAFLSFCIIEIIVRSCRKKSARPAAGVGDTGMEDAKAIRGSASQGPLTESWKDGNPLKKPPPAVAPSSGEEGELHYATLSFHKVKPQDPQGQEATDSEYSEIKIHKRETAETQACLRNHNPSSKEVRG (SEQ ID NO: 155) HumanNM_033130.4 MDGRFWIRVQESVMVPEGLCISVPCSFSYPRQDWTGSTPAYGYWFKAVTETTKGAPSiglec-10 VATNHQSREVEMSTRGRFQLTGDPAKGNCSLVIRDAQMQDESQYFFRVERGSYVRYNFMNDGFFLKVTALTQKPDVYIPETLEPGQPVTVICVFNWAFEECPPPSFSWTGAALSSQGTKPTTSHFSVLSFTPRPQDHNTDLTCHVDFSRKGVSVQRTVRLRVAYAPRDLVISISRDNTPALEPQPQGNVPYLEAQKGQFLRLLCAADSQPPATLSWVLQNRVLSSSHPWGPRPLGLELPGVKAGDSGRYTCRAENRLGSQQRALDLSVQYPPENLRVMVSQANRTVLENLGNGTSLPVLEGQSLCLVCVTHSSPPARLSWTQRGQVLSPSQPSDPGVLELPRVQVEHEGEFTCHARHPLGSQHVSLSLSVHYSPKLLGPSCSWEAEGLHCSCSSQASPAPSLRWWLGEELLEGNSSQDSFEVTPSSAGPWANSSLSLHGGLSSGLRLRCEAWNVHGAQSGSILQLPDKKGLISTAFSNGAFLGIGITALLFLCLALIIMKILPKRRTQTETPRPRFSRHSTILDYINVVPTAGPLAQKRNQKATPNSPRTPLPPGAPSPESKKNQKKQYQLPSFPEPKSSTQAPESQESQEELHYATLNFPGVRPRPEARMPKGTQADYAEVKFQ (SEQ ID NO: 156) HumanNM_052884.2 NKDPSYSLQVQRQVPVPEGLCVIVSCNLSYPRDGWDESTAAYGYWFKGRTSPKTGAPSiglec-11 VATNNQSREVEMSTRDRFQLTGDPGKGSCSLVIRDAQREDEAWYFFRVERGSRVRHSFLSNAFFLKVTALTKKPDVYIPETLEPGQPVTVICVFNWAFKKCPAPSFSWTGAALSPRRTRPSTSHFSVLSFTPSPQDHDTDLTCHVDFSRKGVSAQRTVRLRVAYAPKDLIISISHDNTSALELQGNVIYLEVQKGQFLRLLCAADSQPPATLSWVLQDRVLSSSHPWGPRTLGLELRGVRAGDSGRYTCRAENRLGSQQQALDLSVQYPPENLRVMVSQANRTVLENLGNGTSLPVLEGQSLRLVCVTHSSPPARLSWTRWGQTVGPSQPSDPGVLELPPIQMEHEGEFTCHAQHPLGSQHVSLSLSVHYPPQLLGPSCSWEAEGLHCSCSSQASPAPSLRWWLGEELLEGNSSQGSFEVTPSSAGPWANSSLSLHGGLSSGLRLRCKAWNVHGAQSGSVFQLLPGKLEHGGGLGLGAALGAGVAALLAFCSCLVVFRVKICRKEARKRAAAEQDVPSTLGPISQGHQHECSAGSSQDHPPPGAATYTPGKGEEQELHYASLSFQGLRLWEPADQEAPSTTEYSEIKIHTGQPLRGPGFGLQLEREMSGMVPK (SEQ ID NO: 157) HumanNM_053003.3 KEQKDYLLTMQKSVTVQEGLCVSVLCSFSYPQNGWTASDPVHGYWFRAGDHVSRNISiglec-12 PVATNNPARAVQEETRDRFHLLGDPQNKDCTLSIRDTRESDAGTYVFCVERGNMKWNYKYDQLSVNVTASQDLLSRYRLEVPESVTVQEGLCVSVPCSVLYPHYNWTASSPVYGSWFKEGADIPWDIPVATNTPSGKVQEDTHGRFLLLGDPQTNNCSLSIRDARKGDSGKYYFQVERGSRKWNYIYDKLSVHVTALTHMPTFSIPGTLESGHPRNLTCSVPWACEQGTPPTITWMGASVSSLDPTITRSSMLSLIPQPQDHGTSLTCQVTLPGAGVTMTRAVRLNISYPPQNLTMTVFQGDGTASTTLRNGSALSVLEGQSLHLVCAVDSNPPARLSWTWGSLTLSPSQSSNLGVLELPRVHVKDEGEFTCRAQNPLGSQHISLSLSLQNEYTGKMRPISGVTLGAFGGAGATALVFLYFCIIFVVVRSCRKKSARPAVGVGDTGMEDANAVRGSASQGPLIESPADDSPPHHAPPALATPSPEEGEIQYASLSFHKARPQYPQEQEAIGYEYSEINIPK (SEQ ID NO: 158) Cynomolgus XM_005590087.1QRNNQKNYPLTMQESVTVQQGLCVHVLCSFSYPWYGWISSDPVHGYWFRAGAHT SiglecDRDAPVATNNPARAVREDTRDRFHLLGDPQTKNCTLSIRDARSSDAGTYFFRVETGKTKWNYKYAPLSVHVTALTHRPNILIPGTLESGCPRNLTCSVPWACEQGTAPMISWMGTSVSPLDPSTTRSSVLTLIPQPQDHGTSLTCQVTFPGASVTTNKTIHLNVSYPPQNLTMTVFQGNDTVSIVLGNGSSVSVPEGPSLRLVCAVDSNPPARLSLSWGGLTLCPSQPSNPGVLELPRVHLRDEEEFTCRAQNLLGSQQVSLNVSLQSKATSGLTQGAVGAGATALVFLS FCVIFVVVP(SEQ ID NO: 159) HumanQTSKLLTMQSSVTVQEGLCVHVPCSFSYPSHGWIYPGPVVHGYWFREGANTDQDAP Siglec-9VATNNPARAVWEETRDRFHLLGDPHTENCTLSIRDARRSDAGRYFFRMEKGSIKWNY K100E/A315EKHHRLSVNVTALTHRPNILIPGTLESGCPQNLTCSVPWACEQGTPPMISWIGTSVSPL alleleDPSTTRSSVLTLIPQPQDHGTSLTCQVTFPGASVTTNKTVHLNVSYPPQNLTMTVFQGDGTVSTVLGNGSSLSLPEGQSLRLVCAVDAVDSNPPARLSLSWRGLTLCPSQPSNPGVLELPWVHLRDEAEFTCRAQNPLGSQQVYLNVSLQSKATSGVTQGVVGGAGATALVFLSFCVIFVVVRSCRKKSARPAAGVGDTGIEDANAVRGSASQGPLTEPWAEDSPPDQPPPASARSSVGEGELQYASLSFQMVKPWDSRGQEATDTEYSEIKIHR (SEQ ID NO: 160) HumanMEWSWVFLFFLSVTTGVHSGKPIPNPLLGLDSTQTSKLLTMQSSVTVQEGLCVHVPC Siglec-9SFSYPSHGWIYPGPVVHGYWFREGANTDQDAPVATNNPARAVWEETRDRFHLLGD N-terminalPHTKNCTLSIRDARRSDAGRYFFRMEKGSIKWNYKHHRLSVNVTAATSGVTQGVVG V-set Ig-likeGAGATALVFLSFCVIFVVVRSCRKKSARPAAGVGDTGIEDANAVRGSASQGPLTEPW domainAEDSPPDQPPPASARSSVGEGELQYASLSFQMVKPWDSRGQEATDTEYSEIKIHR (SEQ ID NO: 161)Human MEWSWVFLFFLSVTTGVHSGKPIPNPLLGLDSTLTHRPNILIPGTLESGCPQNLTCSVPSiglec-9 WACEQGTPPMISWIGTSVSPLDPSTTRSSVLTLIPQPQDHGTSLTCQVTFPGASVTTNIg-like KTVHLNVSYPPQNLTMTVFQGDGTVATSGVTQGVVGGAGATALVFLSFCVIFVVVRSC2-type CRKKSARPAAGVGDTGIEDANAVRGSASQGPLTEPWAEDSPPDQPPPASARSSVGEdomain 1 GELQYASLSFQMVKPWDSRGQEATDTEYSEIKIHR (SEQ ID NO: 162) HumanMEWSWVFLFFLSVTTGVHSGKPIPNPLLGLDSTSTVLGNGSSLSLPEGQSLRLVCAVD Siglec-9AVDSNPPARLSLSWRGLTLCPSQPSNPGVLELPWVHLRDAAEFTCRAQNPLGSQQVY Ig-likeLNVSLQSKATSGVTQGVVGGAGATALVFLSFCVIFVVVRSCRKKSARPAAGVGDTGIE vtypeDANAVRGSASQGPLTEPWAEDSPPDQPPPASARSSVGEGELQYASLSFQMVKPWDS domain 2RGQEATDTEYSEIKIHR (SEQ ID NO: 163)

Example 3: Binding to CD33-Related Siglecs

CD33-related Siglecs that share sequence similarity to Siglec-7 and -9are generally divided into two groups, a first subset made up ofSiglec-1, -2, -4 and -15, and the CD33-related group of Siglecs whichincludes Siglec-3, -5, -6, -7, -8, -9, -10, -11, -12, -14 and -16. Sinceother CD33-related Siglecs have different biological functions and/orare not thought to be involved in tumor surveillance, antibodies werefurther screened to assess whether it is possible to obtaincross-reactive Siglec-7/9 antibodies that do not bind to otherCD33-related Siglecs.

Cells expressing Siglec-3, -5, -6, -8, -10, -11 and -12 were generatedand a representative subset of the cross-reactive Siglec-7/9 antibodieswere tested by flow cytometry for binding to the cells. Amino acidsequences and Genbank references for different Siglec used herein areshown below in Table 1, above.

Briefly, HuSiglec-expressing CHO cell lines (that expressed one of theSiglecs) were used. For the flow cytometry screening, antibodies wereincubated 1 hour with each HuSiglec-expressing CHO cell lines (CHOHuSiglec-3 cell line, CHO HuSiglec-5 cell line, CHO HuSiglec-6 cellline, CHO HuSiglec-8 cell line, CHO HuSiglec-10 cell line, CHOHuSiglec-11 cell line, CHO HuSiglec-12 cell line), washed twice instaining buffer, revealed by Goat anti-mouse polyclonal antibody (pAb)labeled with PE, washed twice with staining buffer and stainings wereacquired on a HTFC cytometer and analyzed using the FlowJo software.

Results showed that none of the anti-Siglec-9 antibodies mAbA, mAbB,mAbC, mAbD, mAbE and mAbF bound to any of the Siglecs-3, -5, -6, -7, -8,-10, -11 or -12.

Results showed that some cross-reactive Siglec-7/9 antibodies can becapable of also binding to Siglec-12 or Siglec-6 in addition to Siglec-7and -9. mAb1, mAb2 and mAb3 bound to Siglec-12 in addition to Siglec-7and -9, while mAb3, mAb4, mAb5 and mAb6 did not bind to Siglec-12. Noneof the exemplary antibodies mAb1, mAb2, mAb3, mAb4, mAb5 or mAb6 boundto any of the Siglecs-3, -5, -6, -8, -10, or -11.

Example 4: Titration of Antibodies for Binding to Siglecs

Binding of antibodies on human Siglec-7, human Siglec-9 and CynomolgusSiglec-9 was tested by titration experiment by flow cytometry on CHOcells transfected with human Siglec-7 and human Siglec-9 and CynomolgusSiglec-9. Cells were incubated 1 h in Staining Buffer (SB) with primaryantibodies at 20 ug/ml and a series of dilution of 1:5. They were washedthree times with SB, then incubated 30 min with a Goat F(ab′)²Anti-human IgG (Fc) PE (Beckman Coulter #IM05510), and washed twice withSB. Fluorescence was revealed with HTFC Intellicyt cytometer.

Six antibodies shown below from the 5 fusions in the 2 immunizationswere found to have comparable binding affinity for human Siglec-7 andhuman Siglec-9 as expressed by cells, and furthermore for cynomolgusSiglec. The EC₅₀ values (μg/ml) for binding for each antibody are shownbelow.

mAb1 mAb2 mAb3 mAb4 mAb5 mAb6 EC₅₀ Siglec-7 0.21 0.17 0.22 0.17 0.220.33 (μg/ml) Siglec-9 0.11 0.08 0.23 0.28 0.26 0.31 Siglec-Cyno 0.670.53 0.85 0.17 0.14 0.17

Example 5: Siglec-9 Binding Affinity by Surface Plasmon Resonance (SPR)Biacore™ T100 General Procedure and Reagents

SPR measurements were performed on a Biacore™ T200 apparatus (Biacore™GE Healthcare) at 25° C. In all Biacore™ experiments HBS-EP+(Biacore™ GEHealthcare) and NaOH 10 mM served as running buffer and regenerationbuffer respectively. Sensorgrams were analyzed with Biacore™ T200Evaluation software. Human siglec-9 and -7 multimeric proteins werecloned, produced and purified at Innate Pharma.

Immobilization of Protein-A

Proteins were immobilized covalently to carboxyl groups in the dextranlayer on a Sensor Chip CMS. The chip surface was activated with EDC/NHS(N-ethyl-N′-(3-dimethylaminopropyl) carbodiamide hydrochloride andN-hydroxysuccinimide (Biacore™, GE Healthcare). Proteins were diluted to10 μg/ml in coupling buffer (10 mM acetate, pH 4.2 & 5.0) and injecteduntil the appropriate immobilization level was reached (i.e. 600 to2000RU). Deactivation of the remaining activated groups was performedusing 100 mM ethanolamine pH 8 (Biacore™, GE Healthcare).

Affinity Study

The affinity study was carried out according to a standard Kineticprotocol recommended by the manufacturer (Biacore™ GE Healthcare kineticwizard). Serial dilutions of anti-Siglec-9 and -7/9 antibody Fabfragments ranging from 600 nM to 0.975 nM were sequentially injectedover the immobilized Siglec-9 Fc and Siglec-7 Fc proteins and allowed todissociate for 10 min before regeneration. The entire sensorgram setswere fitted using the 1:1 kinetic binding model. Monovalent affinitiesand kinetic association and dissociation rate constants are shown belowin Table 2 below.

TABLE 2 Fab binding on Siglec-9 Fc protein KD (nM) (1:1 FAB Binding)Koff ⁽¹⁰⁻³⁾ 1/S Fab.A (Fab of mAbA) 0.04  0.025 Fab.B (Fab of mAbB) 0.370.31 Fab.C (Fab of mAbC) 0.55 0.43 Fab.D (Fab of mAbD) 4.12 0.11 Fab.E(Fab of mAbE) 1   1.9  Fab.F (Fab of mAbF) 1   0.46 Fab1 (Fab of mAb1)0.4  0.16 Fab2 (Fab of mAb2) 0.8  0.17 Fab binding on Siglec-7 Fcprotein KD (nM) (1:1 Fab Binding) Koff ⁽¹⁰⁻³⁾ 1/S Fab1 0.06 0.04 Fab20.07 0.04

Example 6: Titration on Monocyte-Derived Dendritic Cells

Generation of Monocyte-Derived Dendritic Cells (moDCs):

Monocyte-derived dendritic cells were generated from peripheral bloodmononuclear cells. PBMCs were isolated from buffy coats, obtained fromhealthy donors. Monocytes were purified using the kit Monocyte IsolationKit II (Miltenyi Biotec) and were differentiated in moDC for a total of6 days in RPMI medium (GIBCO) supplemented with 10% inactivated FBS(GIBCO), Glutamine (GIBCO), MEM NEAA (GIBCO), Sodium pyruvate (GIBCO),IL-4 (20 ng/ml)(Peprotech) and GM-CSF (400 ng/ml)(Miltenyi Biotec).Cells were cultured in a humidified CO2 incubator at 37° C. and thecytokines were renewed on day 4.

moDC were desialylated for 2 hours with 25 mU neuraminidase (RocheDiagnostics). Desialylation was controlled before and afterneuraminidase treatment: moDCs cells were incubated 1 h in StainingBuffer (SB) with mouse Siglec-7 Fc (IPH) and mouse Siglec-9 Fcrecombinant protein (IPH) at 10 ug/ml, washed twice with SB, incubated30 min with a Goat F(ab′)2 Anti-Mouse IgG (Fc) PE (JacksonImmunoResearch), washed twice with SB, and fluorescence was revealedwith Canto II (HTS).

Titrations

Binding on moDCs and neuraminidase treated moDCs was tested in atitration experiment by flow cytometry. Cells were incubated 1 h inStaining Buffer (SB) with primary antibodies at 10 ug/ml and a series ofdilution of 1:10. They were washed two times with SB, then incubated 30min with a Goat F(ab′)² Anti-Human IgG (Fc) PE (Jackson lmmunoresearch),and washed twice with SB. Fluorescence was revealed with HTFC Intellicytcytometer.

Results

The EC₅₀ were highly enhanced (10 fold) after neuraminidase treatment,suggesting that Siglec-9 expressed on moDCs were engaged in cisinteraction with their sialic acid ligands before neuraminidasetreatment. However, the plateau phase level is not modified, suggestingthan the high affinity antibodies can bind all Siglec-9 (bound andunbound) conformations on cell surface and inhibits cis-interactions andsignalling in monoDCs, as well as in other cell types (e.g., NK cells,CD8 T cells, monocytes and macrophages M1 and M2). Results are shown inFIG. 3 for representative antibodies mAbA, mAbC and mAbD in moDC (lefthand panel) and neuramidase-treated moDC (right hand panel), accompaniedby their respective EC₅₀ values.

Example 7: Evaluation of Ability of Antibodies to Neutralize SiglecActivity in NK Cells

Anti-Siglec-7/9 antibodies tested in the first and second immunizationswere tested for blockade of Siglec activity in an NK cell activationassay using primary NK cells (fresh NK cells purified from human donors,incubated overnight at 37° C. before use). Increase of CD137 expressionin 24 hours is correlated with the activation of several lymphocytesincluding NK cells (Kohrt et al. (2011) Blood 117(8):2423-2432). Theeffect of anti-Siglec-7/9 antibody and desialylation of target cells onNK cells activation was determined by analysis of CD137 expression on NKcells by flow cytometry. Each of the anti-Siglec-7/9 mAbs mAb1, mAb2,mAb3, mAb4, mAb5 and mAb6 induced an increase of CD137 expression at 24hours.

The effects of anti-Siglec-7/9 antibodies was then studied bycytotoxicity assays (Cr⁵¹) with YTS Siglec-9* effector cell line (thehuman NK cell line YTS transfected with human Siglec-9) as effector andRamos cell line as target. This test measures the cytotoxicity of YTSSiglec-9* cell line by directly quantifying the lysis of ⁵¹Cr-loadedtarget cells. Briefly, target cells are first labeled with radioactive⁵¹Cr isotope and then co-incubated for 4 h at 37° C. with effectorcells. During this time, target cells that are sensitive to YTS cellsare lysed releasing ⁵¹Cr into the medium. The ⁵¹Cr in the recoveredsupernatant is measured by liquid scintillation counting. The resultsobtained allow evaluating the percent lysis of target cells by NK cells.The assay was carried out in 96 U well plates in completed RPMI, 200 μLfinal/well, with an E:T ratio 5/1. Anti-Siglec-7/9 antibodies andisotype control were added at 10 ug/ml and a series of dilution of 1:10.

Each of the anti-Siglec-7/9 mAbs mAb1, mAb2, mAb3, mAb4, mAb5 and mAb6induced an increase of YTS Siglec-9* cytotoxicity in a dose dependentmanner. As a control, this effect was not observed on wild type YTS cellline (no Siglec-9 expression). Similarly, each of the anti-Siglec-9 mAbsmAbA, mAbB, mAbC, mAbD, mAbE and mAbF induce an increase of YTSSiglec-9* cytotoxicity in a dose dependent manner. FIG. 4 shows dosedependent induction of an increase of YTS Siglec-9* cytotoxicity amongSiglec-7 and -9 cross-reactive antibodies (FIG. 4B) and among theSiglec-9 monospecific (non-Siglec-7 binding) antibodies (FIG. 4A).

Example 8: Detailed Study of Siglec-9 Neutralization in Primary Human NKCells (Low Siglec-9 Expression)

We considered the possibility that the inability of prior antibodies toneutralize Siglec-9 in NK cells might be related to differences inSiglec-9 expression in primary NK cells compared for example toneutrophils and other cells that express much higher levels of Siglec-9at their surface, and Siglec-7 expressed in differing NK cell subsets.In order to investigate whether antibodies could be obtained thatneutralize Siglec-9 in NK cells, we studied and selected antibodies inprimary NK cells from a number of human donors, gated on Siglec-9 byflow cytometry. The effect of anti-Siglec-9 antibodies was studied bycytotoxicity by assessing tumor cell lysis in a classical ⁵¹Cr releaseassay and by activation assays by assessing CD137 surface expression onNK cells. In each case, primary NK cells (as fresh NK cells purifiedfrom donors) were used as effector cells and HT29 colorectal cancer cellline were used as target.

Part 1: Cytotoxicity Assay: Purified NK Vs HT29 Tumor Cells in Two HumanDonors

The cytotoxicity assay measured the cytotoxicity of NK cells by directlyquantifying the lysis of ⁵¹Cr-loaded target cells. Briefly, target cellswere first labeled with radioactive ⁵¹Cr isotope and then co-incubatedfor 4 h at 37° C. with effector cells. During this time, target cellsthat are sensitive to NK cells were lysed releasing ⁵¹Cr into themedium. The ⁵¹Cr in the recovered supernatant were measured by liquidscintillation counting. The results obtained allow the evaluation thepercent lysis of target cells by NK cells. The assay was carried out in96 U well plates in completed RPMI, 200 μL final/well, with an E:T ratio8/1. Anti-Siglec-9 antibodies and isotype control were added at 10ug/ml.

Each of the anti-Siglec9 antibodies mAbA, mAbB, mAbC, mAbD, mAbE, andmAbF and anti-Siglec7/9 antibodies mAb1, mAb2, mAb3, mAb4, mAb5 and mAb6induced an increase of NK cells cytotoxicity. FIG. 5 is a representativefigure showing the increase of primary NK cell cytotoxicity mediated byantibody mAbA, mAbC, mAbD, mAbE, and mAbF in two different human donors(donors D1 (left hand panel) and D2 (right hand panel)).

Part 2: Activation Assay (CD137): Purified NK Vs HT29, mAb Comparison ina Single Human Donor

The effect of the anti-Siglec-7/9 and anti-Siglec-9 antibodies on NKcells activation was determined by analysis of CD137 expression onSiglec-9 positive NK cells by flow cytometry. Effector cells wereprimary NK cells (fresh NK cells purified from donors, incubationovernight at 37° C. before use) and target cells (HT29 cell line) weremixed at a ratio 1:1. The CD137 assay was carried out in 96 U wellplates in completed RPMI, 200 μL final/well. Antibodies werepre-incubated 30 minutes at 37° C. with effector cells and then targetcells were co-incubated overnight at 37° C. The following steps were:spin 3 min at 500 g; wash twice with Staining Buffer (SB); addition of50 μL of staining Ab mix (anti CD3 Pacific blue—BD Pharmingen;anti-CD56-PE-Vio770 (Miltenyi); anti-CD137-APC (Miltenyi), anti Siglec-9K8-PE (Biolegend); incubation 30 min at 4° C.; wash twice with SB;resuspended pellet with SB; and fluorescence revealed with Canto II(HTS).

Negative controls were NK cells vs HT29 alone and in presence of isotypecontrol. FIG. 6 is a representative figure showing the increase of % ofSiglec-9-positive NK cells expressing CD137 mediated by severalanti-Siglec-9 and anti Siglec-7/9 antibodies mAbA, mAbB, mAbF, mAb6 andmAb4 in one human donor. As a control, % of Siglec-9-negative NK cellsexpressing CD137 were not affected by these antibodies. As can be seenin the figure, the anti-Siglec-9 antibodies fully restored cytotoxicityof Siglec-9-expressing primary human NK cells to the level observed inSiglec-9-negative primary human NK cells from the same donor.

Part 3: Activation Assay (CD137): Purified NK Vs HT29, mAbA and mAb1 in6 Human Donors

Experiments were reproduced with 6 donors by using one anti Siglec-9(mAb.A) and one anti Siglec-7/9 (mAb1). In absence of antibodies (the“medium” setting), the % of NK expressing CD137 varied among donorsbetween 6% and 27% (see (FIG. 7, left hand panel)). Data were normalizedto be a relative change compared to the control medium mAb1 induced anincrease of Siglec-9+CD137+NK % (FIG. 7, middle panel) and not Siglec-9−CD137+NK % (FIG. 7, right hand panel).

Example 9: Titration on Primary NK Cells

Binding of antibodies on fresh purified human NK cells was tested bytitration experiment by flow cytometry. Cells were incubated 1 h inStaining Buffer (SB) with primary antibodies at 10 ug/ml and a series ofdilution of 1:10. They were washed three times with SB, then incubated30 min with a Goat F(ab′)² Anti-Human IgG (Fc) PE (JacksonImmunoResearch). Stainings were acquired on a BD FACS Canto II andanalyzed using the FlowJo software. EC₅₀ values are shown in the tablebelow in μg/ml (calculated using a 4-parameter logistic fit).

Mean EC50 (μg/ml) - 4 donors mAb1 0.05 mAb2 0.07 mAb3 0.19 mAb4 0.61mAb5 1.27 mAb6 1.30 mAbA 0.08 mAbB 0.10 mAbC 0.09 mAbE 0.01 mAbF 0.30

Example 10: Blockade of Siglec Binding to Sialic Acid Ligands

Part A: Blockade of Siglec-9 Binding to Sialic Acid Expressing TumorCells by Flow Cytometry

A dose-range of anti-human Siglec-9 Fab were co-incubated 30 minutes atroom temperature with the human Siglec-9 Fc fusion recombinant proteinat a fixed dose, then added on various sialic acid expressing cell linesK562 E6 (K562 cell line transfected with human HLA-E) and Ramos for 1hour. After washing cells two times in staining buffer, a PE-coupledgoat anti-mouse IgG Fc fragment secondary antibodies (Jacksonlmmunoresearch) diluted in staining buffer were added to the cells andplates were incubated for 30 additional minutes at 4° C. Cells werewashed two times and analyzed on an Accury C6 flow cytometer equippedwith an HTFC plate reader. Mean of fluorescence vs. ratio of Fab andSiglec-9 Fc fusion recombinant protein was plotted on graphs.

Results are shown in FIG. 8. On the top panel, shown is binding ofSiglec-9-Fc protein to Ramos cells in the presence of antibodies. Theanti-Siglec/9 mAbs mAbA, mAbB, mAbC, and mAbD each inhibited binding ofSiglec-9-Fc protein to the Ramos cells, while mAbE showed a partialability to inhibit binding of Siglec-9-Fc protein to the Ramos cells,and mAbF did not significantly inhibit binding of Siglec-9-Fc protein tothe Ramos cells. In FIG. 8, bottom panel, shown is binding ofSiglec-9-Fc protein to K562 cells in the presence of antibodies. Theanti-Siglec/9 mAbs mAbA, mAbB, mAbC and mAbD each inhibited binding ofSiglec-9-Fc protein to the Ramos cells, while both mAbE and mAbF showeda partial ability to inhibit binding of Siglec-9-Fc protein to the K562cells, and only at significantly higher concentrations of antibody. Inconclusion, the antibody mAbA, mAbB, mAbC, and mAbD block totally thebinding of Siglec-9 to its sialic acid ligands on tumor cells whileantibodies mAbE blockade depend on sialic acid expressing cell line andmAbF does not block the binding.

Part B: Blockade of Siglec-7 and -9 binding to sialylated ligands byELISA assays Sialic acids are nine-carbon carboxylated monosaccharideson glycosylated proteins and lipids formed. Several enzymes includingsialyltransferases (catalyzing their biosynthesis) and sialidases alsotermed as neuraminidases (catalyzing their cleavage), regulate theiroccurrence in the mammalian system. In cancer, altered sialic acidprofile plays dominant role enhancing tumor growth, metastasis andevading immune surveillance, leading to cancer cell survival (Bork etal., J Pharm Sci. 2009 October; 98(10):3499-508). Increased sialylationstogether with altered enzyme profile regulating sialylation has beenreported in several cancers. The ST3GAL6 enzyme is overexpressed inmultiple myeloma cell lines and patients and is associated in vitro withexpression of α-2,3-linked sialic acid on the surface of multiplemyeloma cells. In vivo, ST3GAL6 knockdown is associated with reducedhoming and engraftment of multiple myeloma cells to the bone marrowniche, along with decreased tumor burden and prolonged survival (Glaveyet al., Blood. 2014 Sep. 11; 124(11):1765-76). High ST3GAL1 enzymeexpression in glioma is associated with higher tumor grades of themesenchymal molecular classification (Chong et al., Natl Cancer Inst.2015 Nov. 7; 108(2). Aberrant promoter methylation play a role inmodulation of several sialyl transferases expression in cancer (Vojta etal., Biochim Biophys Acta. 2016 Jan. 12). In bladder cancer, aberrantST6GAL1 promoter methylation induces ST6Gal1 expression loss (Antony etal., BMC Cancer. 2014 Dec. 2; 14:901).

Siglec-7 and Siglec-9 bind to various sialic acid linkages. A sialosidelibrary printed on chip identified sialoside ligands common to severalSiglec and one selective Siglec-7 ligand (Rillahan et al., ACS ChemBiol. 2013 Jul. 19; 8(7):1417-22). In view of the possible differentialrecognition of sialosides by Siglec-7 and Siglec-9, targeting bothSiglec-7 and -9 on immune cells could allow targeting of several cancertypes given the various sialyl transferases and sialic acid.

Blocking of the interaction between Siglec-7 and -9 and sialylatedligands by anti Siglec-7/9 antibodies was tested on ELISA assays. Siglecproteins were Siglec-7 human Fc and Siglec-9 Human Fc recombinantproteins, and ligands were biotinylated polymers with sialylatedtrisaccharides (Neu5Aca2-3Galb1-4GlcNAcb-PAA-biotin Glycotech #01-077referred to as “Sia1” and 6′-Sialyllactose-PAA-biotin Glycotech #01-039(referred to as “Sia2”). Briefly, Protein A was coated on ELISA platesover night at 4° C. After 3 washes and saturation, Siglec-7 Fc andSiglec-9 Fc were added at 0.8 μg/well at RT for 1H30. After 3 washes,mAbs were added at 20 ug/ml and a series of dilution of 1:5. After 3washes, biotinylated sialylated polymers were added for 3 hours at roomtemperature. After 3 washes, Streptavidin-Peroxidase (Beckman) was addedat 1:1000. Finally, binding of sialylated polymers on Siglec-7 and -9proteins was revealed by addition of TMB (Interchim) at RT in darkness,and the reaction was stopped by addition of H2S04. The absorbance wasread at 450 nm.

Results are shown in FIGS. 9 and 10. mAbs 1, 2, 4, 5 and 6 blockSiglec-7 interaction with Sia2, but mAb3 did not (FIG. 9). All mAbsblocked the Siglec-9 interaction with Sia2 (FIG. 10), however mAb1, mAb2and mAb3 showed low ability to inhibit the Siglec-9 interaction withSia1 (FIG. 10), and thus did not substantially block the Sia1interaction. mAb5 and mAb6 blocked the Siglec-9 interaction with Sia1,and mAb4 had intermediate ability to block the Siglec-9 interaction withSia1. The blocking effect on Siglec-9 is dependent on sialic acid type.On the overall, the most complete inhibition was observed withanti-Siglec-9 antibodies mAbA, mAbB, mAbC and mAbD which achievedsubstantially full inhibition of the Siglec-9 interaction with sialicacids.

Example 11: Epitopes of Anti-Siglec Antibodies Using Point Mutants

In order to define the epitopes of anti-Siglec-9 antibodies, we firstidentified the binding domain of our antibodies by expressing eachsingle Siglec-9 domain (V-set Ig-like domain, Ig-like C2-type domain 1,and Ig-like C2-type domain 2) with the tag V5 in HEK293T cells andtesting binding of the antibodies to each protein.

We then designed Siglec-9 mutants defined by substitutions of aminoacids exposed at the molecular surface over the surface of theN-terminal V-set Ig-like domain. The structure of Siglec-9 has not beenresolved yet, and among available Siglec structures, Siglec-7 is theclosest member (more than 80% of identity with Siglec-9 amino acidsequence). Consequently, we used the Siglec-7 structure to designSiglec-9 mutants. The native Siglec-9 peptide leader of the polypeptideof SEQ ID NO: 2 was replaced by a substitute leader sequence and V5 tag(shown in the Siglec-9 domain proteins V-set Ig-like domain, Ig-like02-type domain 1, and Ig-like 02-type domain 2 in Table 1), followed bythe Siglec-9 amino acid sequence of Table 1, into which wereincorporated amino acid substitutions listed in Table 3. Proteins wereexpressed in the HEK293T cell line.

All figures (FIGS. 11-14) correspond to the N-terminal V-set Ig-likedomain of SIGLEC-7 structure 107V, described by Alphey et al (2003),supra. The figures show in light shading the ligand binding area,including arginine 124, which is a key residue conserved in all Siglecsfor the interaction with the carboxyl group on the terminal sialic acidsugar, and surrounding residues W132, K131, K135 and N133 which areconserved between Siglec-7 and Siglec-9 and are also described asessential for sialic acid binding. W132 provides a hydrophobicinteraction with the glycerol moiety of sialic acid. The targeted aminoacid mutations in the Table 3 are residues present in both Siglec-7 and-9, and are shown using numbering of SEQ ID NO: 1 for Siglec-7 or SEQ IDNO: 2 for Siglec-9 (residue in wild type Siglec-9/position ofresidue/residue in mutant).

TABLE 3 Mutations with Reference to Mutations with Reference to Ref.Siglec-7 of SEQ ID NO: 1 Siglec-9 of SEQ ID NO: 2 M1 Q19A-T20A-S21N-K22AQ18A-T19A-S20N-K21A M2 L27T-T29A-S47A-S49A-K104NL22T-T24A-S42A-S44A-K100N M3 Q31E-S33K-T35V Q26E-S28K-T30V M5H43L-P45A-H96F-L98S-N105D-T107A- H38L-P40A-H92F-L94S-N101D- S109AT103A-S105A M6 S52L-H53T-G54D-W55S-I56A-Y57A-S47L-H48T-G49D-W50S-I51A-Y52A- P58A-G59S P53A-G54S M7P60S-H62A-E126A-G128S-S129K- P55S-H58A-E122A-G124S-S125K- K131A K127A M8R67A-A70T-N71A-T72R-D73R-Q74K- R63A-A66T-N67A-T68R-D69R-Q70K- D75A D71AM9 N82A-P83S-A84S-R85S-A86K-V87S N78A-P79S-A80S-R81S-A82K-V83S M10N81A-D100A-H102W-T103R N77A-D96A-H98W-T99R M11 W88V-E89K-E90A-R92AW84V-E85K-E86A-R88A M12 D93A-R94A-R111S-D112A-R114AD89A-R90A-R107S-D108A-R110A M13 E38A-R115A-S116K-N142V-T144A-E33A-R111A-S112K-N138V-T140A- A118S A114S M14 R124A-W132Y-N133AR120A-W128Y-N129A M15 H137D-R138A-R120S-S32R H133D-R134A-R116S-S27R M16K135M-H136W K131M-H132W

Generation of Mutants

Siglec-9 mutants were generated by PCR. The sequences amplified were runon agarose gel and purified using the Macherey Nagel PCR Clean-Up GelExtraction kit. The purified PCR products generated for each mutant werethen ligated into an expression vector, with the ClonTech InFusion™system. The vectors containing the mutated sequences were prepared asMiniprep and sequenced. After sequencing, the vectors containing themutated sequences were prepared as Midiprep™ using the PromegaPureYield™ Plasmid Midiprep System. HEK293T cells were grown in DMEMmedium (Invitrogen), transfected with vectors using Invitrogen'sLipofectamine™ 2000 and incubated at 37° C. in a CO² incubator for 24 or48 hours prior to testing for transgene expression.

Flow Cytometry Analysis of Anti-Siglec-9 Binding to the HEK293TTransfected Cells

Antibodies mAb4, mAb5 and mAb6 bound the Ig-like C2-type domain 1whereas mAbA, mAbB, mAbC, mAbD, mAbE mAbF, mAb1, mAb2 and mAb3 bound theN-terminal V-set Ig-like domain. The V-set Ig-like domain bindingantibodies were tested for their binding to each of mutants 1-16 by flowcytometry. A first experiment was performed to determine antibodies thatlose their binding to one or several mutants at one concentration. Toconfirm a loss of binding, titration of antibodies was done onantibodies for which binding seemed to be affected by the Siglec-9mutations. Results are shown in Table 4, below.

No antibodies lost binding to mutant M2 which include a substation atresidue K100 (with reference to Siglec-9 of SEQ ID NO: 2) or K104 (withreference to Siglec-7 of SEQ ID NO: 1) that varies in the population;thus the antibodies will bind to the Siglec-9 allele shown in Table 1(SEQ ID NO: 160).

The anti-Siglec-7 and -9 specific antibodies mAb1, mAb2 and mAb3, andthe Siglec-9 specific antibodies mAbE and mAbF all lost binding tomutants M9, M10 and M11 of Siglec-9, but not to any other mutant. Mutant9 contains amino acid substitutions at residues N78, P79, A80, R81, A82and V83 (reference to Siglec-9), indicating that one or more, or all of,the residues of the mutant are important to the core epitope of theseantibodies. Mutant 10 contains amino acid substitutions at residues N77,D96, H98 and T99, indicating that one or more, or all of, the residuesof the mutant are important to the core epitope of these antibodies.Mutant 11 contains amino acid substitutions at residues W84, E85, E86and R88, indicating that one or more, or all of, the residues of themutant are important to the core epitope of these antibodies. As shownin FIG. 11, the residues substituted in M9, M10 and M11 are found on theside of the N-terminal V-set Ig-like domain (dark shading), away fromthe face that contains the sialic acid binding sites (light shading).Notably, the antibodies did not lose binding to M8 which has mutationsin the C—C′ loop domain which defines the sialic ligand specificity ofSiglecs (see, e.g., Alphey et al., 2003 J. Biol. Chem.278(5):3372-3377), nor to M15 of M16 which cover in part a ligandbinding region. The antibodies therefore achieve high potency inblocking Siglec-9, without binding to a sialic acid contact region orbinding site, or to the C—C′ loop.

The anti-Siglec-9 specific antibody mAbD lost binding to mutant M6, butnot to any other mutant. Mutant 6 contains amino acid substitutions atresidues S47, H48, G49, W50, 151, Y52, P53 and G54 (reference toSiglec-9), indicating that one or more, or all of, the residues of themutant are important to the core epitope of the antibody. As shown inFIG. 12, the residues substituted in M6 (dark shading) are found on thetop of the N-terminal V-set Ig-like domain face that contains the sialicacid binding sites, but outside the ligand binding site (light shading).mAbD did not lose binding to M7, but did show partial decrease inbinding to this mutant M7; M7 contains residues that may partiallyoverlap into the ligand binding region (in light shading). M7 includedamino acid substitutions at residues P55, H58, E122, G124, S125 and K127(reference to Siglec-9). Thus, while the residues of M7 are notimportant to the core epitope of the antibody. The antibodies did notlose binding to M8 which has mutations in the C—C′ loop domain or to M15of M16 which cover in part a ligand binding region. The antibodiestherefore achieve high potency in blocking Siglec-9, without binding toa sialic acid contact region or binding site, or to the C—C′ loop.

The anti-Siglec-9 specific antibodies mAbA and mAbB both lost binding tomutant M16 of Siglec-9, but not to any other mutant. Mutant 16 containsamino acid substitutions at residues K131 and H136 (reference toSiglec-9), indicating that one or more, or all of, the residues of themutant are important to the core epitope of these antibodies.Interestingly, while M16 is proximal or within a sialic acid ligandcontact site of Siglec-9 (see FIG. 13), the antibodies did not losebinding to M8 (C—C′ loop domain mutant, nor to M15. The antibodiestherefore achieve high potency in blocking Siglec-9, and moreover withina sialic acid contact region, yet without binding to the C—C′ loop.

Antibody mAbC on the other hand lost binding to mutant M8 of Siglec-9(i.e. within the C—C′ loop) but did not lose binding to M15 or M16, norto M6, M7 or M8, nor to M9, M10 or M11 (nor to any other mutant),although a partial decrease in binding to M15 and M16. The residuesmutated in M8 are shown in FIG. 14. Mutant 8 contains amino acidsubstitutions at residues R63, A66, N67, T68, D69, Q70 and D71(reference to Siglec-9), indicating that one or more, or all of, theresidues of the mutant are important to the core epitope of theseantibodies. The antibody thus binds to residues in the C—C′ loop domainthat defines the sialic acid specificity of Siglecs.

TABLE 4 MUTANT → M1 M2 M3 M5 M6 M7 M8 M9 M10 M11 M14 M15 M16 ANTIBODYmAb1 + + + + + + + − − − + + + mAb2 + + + + + + + − − − + + +mAb3 + + + + + + + − − − + + + mAb.A + + + + + + + + + + + + −mAb.B + + + + + + + + + + + + − mAb.C + + + + + + − + + + + +/− +/−mAb.D + + + + − +/− + + + + + + + mAb.E + + + + + + + − − − + + +mAb.F + + + + + + + − − − + + +

All references, including publications, patent applications, andpatents, cited herein are hereby incorporated by reference in theirentirety and to the same extent as if each reference were individuallyand specifically indicated to be incorporated by reference and were setforth in its entirety herein (to the maximum extent permitted by law),regardless of any separately provided incorporation of particulardocuments made elsewhere herein.

The use of the terms “a” and “an” and “the” and similar referents in thecontext of describing the invention are to be construed to cover boththe singular and the plural, unless otherwise indicated herein orclearly contradicted by context.

Unless otherwise stated, all exact values provided herein arerepresentative of corresponding approximate values (e.g., all exactexemplary values provided with respect to a particular factor ormeasurement can be considered to also provide a correspondingapproximate measurement, modified by “about,” where appropriate).

The description herein of any aspect or embodiment of the inventionusing terms such as “comprising”, “having,” “including,” or “containing”with reference to an element or elements is intended to provide supportfor a similar aspect or embodiment of the invention that “consists of”,“consists essentially of”, or “substantially comprises” that particularelement or elements, unless otherwise stated or clearly contradicted bycontext (e.g., a composition described herein as comprising a particularelement should be understood as also describing a composition consistingof that element, unless otherwise stated or clearly contradicted bycontext).

The use of any and all examples, or exemplary language (e.g., “such as”)provided herein, is intended merely to better illuminate the inventionand does not pose a limitation on the scope of the invention unlessotherwise claimed. No language in the specification should be construedas indicating any non-claimed element as essential to the practice ofthe invention.

We claim:
 1. An in vitro method for modulating the activity ofmonocyte-derived cells and/or lymphocytes, optionally CD56dim NK cells,CD56bright NK cell and/or CD8+ T cells, the method comprising bringingmonocyte-derived cells and/or lymphocytes expressing Siglec-9 intocontact with an antibody that binds a human Siglec-9 polypeptide and iscapable of neutralizing the inhibitory activity of a Siglec-9polypeptide expressed by a human NK cell, wherein the antibody isselected from the group consisting of: (a) an antibody comprising (i) aheavy chain variable region comprising CDR 1, 2 and 3 of the heavy chainvariable region of SEQ ID NO: 15 and (ii) a light chain variable regioncomprising CDR 1, 2 and 3 of the light chain variable region of SEQ IDNO: 16; (b) an antibody comprising (i) a heavy chain variable regioncomprising CDR 1, 2 and 3 of the heavy chain variable region of SEQ IDNO: 17 and (ii) a light chain variable region comprising CDR 1, 2 and 3of the light chain variable region of SEQ ID NO: 18; (c) an antibodycomprising (i) a heavy chain variable region comprising CDR 1, 2 and 3of the heavy chain variable region of SEQ ID NO: 19 and (ii) a lightchain variable region comprising CDR 1, 2 and 3 of the light chainvariable region of SEQ ID NO: 20; (d) an antibody comprising (i) a heavychain variable region comprising CDR 1, 2 and 3 of the heavy chainvariable region of SEQ ID NO: 21 and (ii) a light chain variable regioncomprising CDR 1, 2 and 3 of the light chain variable region of SEQ IDNO: 22; (e) an antibody comprising (i) a heavy chain variable regioncomprising CDR 1, 2 and 3 of the heavy chain variable region of SEQ IDNO: 23 and (ii) a light chain variable region comprising CDR 1, 2 and 3of the light chain variable region of SEQ ID NO: 24; and (f) an antibodycomprising (i) a heavy chain variable region comprising CDR 1, 2 and 3of the heavy chain variable region of SEQ ID NO: 25 and (ii) a lightchain variable region comprising CDR 1, 2 and 3 of the light chainvariable region of SEQ ID NO:
 26. 2. The method of claim 1, wherein thecells are present in a biological sample obtained from a subject havinga disease, optionally a cancer.
 3. An in vitro method for assessing theactivity of NK cells from a subject having disease, the methodcomprising: (i) obtaining a biological sample from a subject comprisingNK cells, bringing said cells into contact with an antibody that binds ahuman Siglec-9 polypeptide and is capable of neutralizing the inhibitoryactivity of a Siglec-9 polypeptide expressed by a human NK cell, whereinthe antibody is selected from the group consisting of: (a) an antibodycomprising (i) a heavy chain variable region comprising CDR 1, 2 and 3of the heavy chain variable region of SEQ ID NO: 15 and (ii) a lightchain variable region comprising CDR 1, 2 and 3 of the light chainvariable region of SEQ ID NO: 16; (b) an antibody comprising (i) a heavychain variable region comprising CDR 1, 2 and 3 of the heavy chainvariable region of SEQ ID NO: 17 and (ii) a light chain variable regioncomprising CDR 1, 2 and 3 of the light chain variable region of SEQ IDNO: 18; (c) an antibody comprising (i) a heavy chain variable regioncomprising CDR 1, 2 and 3 of the heavy chain variable region of SEQ IDNO: 19 and (ii) a light chain variable region comprising CDR 1, 2 and 3of the light chain variable region of SEQ ID NO: 20; (d) an antibodycomprising (i) a heavy chain variable region comprising CDR 1, 2 and 3of the heavy chain variable region of SEQ ID NO: 21 and (ii) a lightchain variable region comprising CDR 1, 2 and 3 of the light chainvariable region of SEQ ID NO: 22; (e) an antibody comprising (i) a heavychain variable region comprising CDR 1, 2 and 3 of the heavy chainvariable region of SEQ ID NO: 23 and (ii) a light chain variable regioncomprising CDR 1, 2 and 3 of the light chain variable region of SEQ IDNO: 24; and (f) an antibody comprising (i) a heavy chain variable regioncomprising CDR 1, 2 and 3 of the heavy chain variable region of SEQ IDNO: 25 and (ii) a light chain variable region comprising CDR 1, 2 and 3of the light chain variable region of SEQ ID NO: 26; and (ii) assessingwhether the antibody modulate the activity of the NK cells.
 4. A methodfor selecting subjects having a cancer that responds to a treatment withan antibody that binds a human Siglec-9 polypeptide and is capable ofneutralizing the inhibitory activity of a Siglec-9 polypeptide expressedby a human NK cell, wherein the antibody is selected from the groupconsisting of: (a) an antibody comprising (i) a heavy chain variableregion comprising CDR 1, 2 and 3 of the heavy chain variable region ofSEQ ID NO: 15 and (ii) a light chain variable region comprising CDR 1, 2and 3 of the light chain variable region of SEQ ID NO: 16; (b) anantibody comprising (i) a heavy chain variable region comprising CDR 1,2 and 3 of the heavy chain variable region of SEQ ID NO: 17 and (ii) alight chain variable region comprising CDR 1, 2 and 3 of the light chainvariable region of SEQ ID NO: 18; (c) an antibody comprising (i) a heavychain variable region comprising CDR 1, 2 and 3 of the heavy chainvariable region of SEQ ID NO: 19 and (ii) a light chain variable regioncomprising CDR 1, 2 and 3 of the light chain variable region of SEQ IDNO: 20; (d) an antibody comprising (i) a heavy chain variable regioncomprising CDR 1, 2 and 3 of the heavy chain variable region of SEQ IDNO: 21 and (ii) a light chain variable region comprising CDR 1, 2 and 3of the light chain variable region of SEQ ID NO: 22; (e) an antibodycomprising (i) a heavy chain variable region comprising CDR 1, 2 and 3of the heavy chain variable region of SEQ ID NO: 23 and (ii) a lightchain variable region comprising CDR 1, 2 and 3 of the light chainvariable region of SEQ ID NO: 24; and (f) an antibody comprising (i) aheavy chain variable region comprising CDR 1, 2 and 3 of the heavy chainvariable region of SEQ ID NO: 25 and (ii) a light chain variable regioncomprising CDR 1, 2 and 3 of the light chain variable region of SEQ IDNO: 26; the method comprising determining whether cancer cells in saidsubject express a ligand of Siglec-9, the expression of sialic acidligand(s) of Siglec-9 or elevated levels of sialic acid ligand(s) ofSiglec-9, being indicative of a responder subject.
 5. The method ofclaim 4, further comprising administering to a responder subject anantibody that binds a human Siglec-9 polypeptide and is capable ofneutralizing the inhibitory activity of a Siglec-9 polypeptide expressedby a human NK cell, wherein the antibody is selected from the groupconsisting of: (a) an antibody comprising (i) a heavy chain variableregion comprising CDR 1, 2 and 3 of the heavy chain variable region ofSEQ ID NO: 15 and (ii) a light chain variable region comprising CDR 1, 2and 3 of the light chain variable region of SEQ ID NO: 16; (b) anantibody comprising (i) a heavy chain variable region comprising CDR 1,2 and 3 of the heavy chain variable region of SEQ ID NO: 17 and (ii) alight chain variable region comprising CDR 1, 2 and 3 of the light chainvariable region of SEQ ID NO: 18; (c) an antibody comprising (i) a heavychain variable region comprising CDR 1, 2 and 3 of the heavy chainvariable region of SEQ ID NO: 19 and (ii) a light chain variable regioncomprising CDR 1, 2 and 3 of the light chain variable region of SEQ IDNO: 20; (d) an antibody comprising (i) a heavy chain variable regioncomprising CDR 1, 2 and 3 of the heavy chain variable region of SEQ IDNO: 21 and (ii) a light chain variable region comprising CDR 1, 2 and 3of the light chain variable region of SEQ ID NO: 22; (e) an antibodycomprising (i) a heavy chain variable region comprising CDR 1, 2 and 3of the heavy chain variable region of SEQ ID NO: 23 and (ii) a lightchain variable region comprising CDR 1, 2 and 3 of the light chainvariable region of SEQ ID NO: 24; and (f) an antibody comprising (i) aheavy chain variable region comprising CDR 1, 2 and 3 of the heavy chainvariable region of SEQ ID NO: 25 and (ii) a light chain variable regioncomprising CDR 1, 2 and 3 of the light chain variable region of SEQ IDNO:
 26. 6. A method of producing an antibody which neutralizes theinhibitory activity of a Siglec-9 polypeptide, comprising: (a) providinga plurality of antibodies that bind a Siglec-9 polypeptide, (b)selecting antibodies (e.g., those of step of (a)) that neutralize theinhibitory activity of a Siglec-9 polypeptide, and (c) selectingantibodies (e.g., those of step (b)) that substantially block theinteraction between a Siglec-9 polypeptide and a sialic acid ligandthereof, wherein the sialic acid ligand comprises aNeu5Aca2-3Galb1-4GlcNAcb structure.
 7. The method of claim 6, whereinassessing ability to neutralize the inhibitory activity of a Siglecpolypeptide comprises selecting an antibody capable of causing anincrease in cytotoxicity, optionally a marker of cytotoxicity, in NKcells purified from human donors that express Siglec-9, when the NKcells are brought into contact with a target human cell bearing a ligandof the Siglec on the target cell surface.
 8. An antibody or antibodyfragment that binds a human Siglec-9 polypeptide, comprising: a heavychain CDR1 comprising the amino acid sequence SYWMH (SEQ ID NO: 75); aheavy chain CDR2 comprising the amino acid sequence EINPSNGHTNYNEKFES(SEQ ID NO: 78); a heavy chain CDR3 comprising the amino acid sequenceGVESYDFDDALDY (SEQ ID NO: 80); a light chain CDR1 comprising the aminoacid sequence RASQDINNYLN (SEQ ID NO: 83); a light chain CDR2 comprisingthe amino acid sequence YTSRLHS (SEQ ID NO: 58); a light chain CDR3comprising the amino acid sequence QQGNTLPFT (SEQ ID NO: 86); and humanheavy and light chain framework sequences.
 9. The antibody of claim 8,wherein the antibody is an antibody having an Fc domain that is modifiedto reduce binding between the Fc domain and an Fcγ receptor.
 10. Theantibody of claim 8, wherein said antibody is an antibody fragment. 11.An isolated Siglec binding protein comprising an antibody fragment ofclaim
 10. 12. A pharmaceutical composition comprising an antibody orantibody fragment according to claim 8, and a pharmaceuticallyacceptable carrier.
 13. A kit comprising the antibody or antibodyfragment of claim 8 and a labelled secondary anti-IgG antibody thatspecifically recognizes said antibody.
 14. A nucleic acid encoding aheavy and/or light chain of an antibody or antibody fragment of claim 8.15. A hybridoma or recombinant host cell producing the antibody orantibody fragment of claim
 8. 16. A method for the treatment of a cancerin a patient in need thereof, the method comprising administering tosaid patient an effective amount of an antibody or antibody fragment ofclaim
 8. 17. An antibody or antibody fragment that binds a humanSiglec-9 polypeptide, comprising: a heavy chain CDR1 comprising theamino acid sequence DYSMH (SEQ ID NO: 112); a heavy chain CDR2comprising the amino acid sequence WIITETGEPTYADDFRG (SEQ ID NO: 115); aheavy chain CDR3 comprising the amino acid sequence DFDGY (SEQ ID NO:117); a light chain CDR1 comprising the amino acid sequence RASENIYSYLA(SEQ ID NO: 119); a light chain CDR2 comprising the amino acid sequenceNAKTLTE (SEQ ID NO: 122); a light chain CDR3 comprising the amino acidsequence QHHYGFPWT (SEQ ID NO: 123); and human heavy and light chainframework sequences.
 18. The antibody of claim 17, wherein the antibodyis an antibody having an Fc domain that is modified to reduce bindingbetween the Fc domain and an Fcγ receptor.
 19. The antibody of claim 17,wherein said antibody is an antibody fragment.
 20. An isolated Siglecbinding protein comprising an antibody fragment of claim
 19. 21. Apharmaceutical composition comprising an antibody or antibody fragmentaccording to claim 17, and a pharmaceutically acceptable carrier.
 22. Akit comprising the antibody or antibody fragment of claim 17 and alabelled secondary anti-IgG antibody that specifically recognizes saidantibody.
 23. A nucleic acid encoding a heavy and/or light chain of anantibody or antibody fragment of claim
 17. 24. A hybridoma orrecombinant host cell producing the antibody or antibody fragment ofclaim
 17. 25. A method for the treatment of a cancer in a patient inneed thereof, the method comprising administering to said patient aneffective amount of an antibody or antibody fragment of claim 17.